Compositions and methods for detecting and treating prostate carcinoma

ABSTRACT

Compositions and methods for the diagnosis, treatment and prevention of prostate cancer, as well as for treatment selection.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the following U.S. ProvisionalApplication Ser. No. 61/246,356, filed Sep. 28, 2009; the entirecontents of which are incorporated herein by this reference. Thisapplication may be related to International Patent Application Nos.:PCT/US2008/059966, filed Apr. 10, 2008, and PCT/US2009/002268, filedApr. 10, 2009, which claims the benefit of U.S. Provisional ApplicationSer. No. 61/123,867, the disclosures of which are hereby incorporatedherein in their entireties by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH

This work was supported by the following grant from the NationalInstitutes of Health, Grant No: HL-70143. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Prostate cancer is a leading healthcare concern in North America andEurope. There were an estimated 232,090 new cases of prostate cancerdiagnosed in 2005 in the United States, and over 30,350 deaths fromadvanced metastatic disease. Prostate cancer is now the most commonlydiagnosed lethal malignancy, and the second leading cause of cancerdeath of men in the United States. Although curative treatment (e.g.,radical prostatectomy or radiotherapy) is feasible for many patientswith the earliest stage disease, a subset of patients have prostatecancer that is resistant to conventional treatments, that is locallyadvanced, or that is metastatic. Metastatic prostate cancer is initiallytreated with androgen deprivation, which achieves stabilization orregression of disease in more than 80% of patients. Nevertheless, allpatients with metastatic prostate cancer ultimately develop androgenresistant disease. The median survival for such patients isapproximately one year. Treatment recommendations for subjects withmetastatic prostate cancers include experimental therapy conducted inthe setting of peer reviewed clinical trials, underscoring the fact thatcurrent standard therapies are inadequate and new approaches oftreatment are needed.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions andmethods for the diagnosis, treatment and prevention of a variety ofneoplasias, including prostate cancer, as well as for treatmentselection.

In one aspect, the invention provides a method for identifying ordiagnosing a neoplasia (e.g., prostate cancer) in a subject, the methodcomprising identifying or detecting an increased level of a nucleic acidmolecule or polypeptide Marker selected from any one or more of OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR in a biological samplederived from the subject, relative to the level present in a reference,thereby identifying or diagnosing the subject as having neoplasia orprostate cancer. In specific embodiments, the markers used are OCT3/4,Nanog, and Sox2. In other specific embodiments, the markers used areOCT3/4, Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a method for identifying ordiagnosing a neoplasia (e.g., prostate cancer, metastatic prostatecancer or prostate cancer having a propensity to metastasize), themethod comprising comparing the level of a nucleic acid molecule orpolypeptide Marker selected from any one or more of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and uPAR in a biological sample, relative to thelevel present in a reference, wherein an increase in the level of one ormore of said Markers identifies or diagnoses the neoplasia or prostatecancer as metastatic or as having a propensity to metastasize. In oneembodiment, the absence of an increase in the level of one or moreMarkers identifies or diagnoses the neoplasia or prostate cancer asnon-metastatic or as lacking the propensity to metastasize. In anotherembodiment, the absence of an increase in the level of Sox2 identifiesor diagnoses the neoplasia or prostate cancer as non-metastatic or aslacking the propensity to metastasize. In specific embodiments, themarkers used are OCT3/4, Nanog, and Sox2. In other specific embodiments,the markers used are OCT3/4, Nanog, Sox2, and c-Myc.

In yet another aspect, the invention provides a method for identifyingor diagnosing a subject as having or having a propensity to develop aneoplasia or metastatic prostate carcinoma, the method comprisingcomparing the level of a nucleic acid molecule or polypeptide Markerselected from any one or more of OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, and uPAR in a biological sample derived from the subjectrelative to the level present in a reference, wherein an increase in thelevel of one or more of said Markers identifies or diagnoses theneoplasia or prostate cancer as metastatic or as having a propensity tometastasize. In one embodiment, the absence of an increase in the levelof one or more Markers identifies or diagnoses the neoplasia or prostatecancer as non-metastatic or as lacking the propensity to metastasize. Inanother embodiment, the absence of an increase in the level of Sox2identifies or diagnoses the neoplasia or prostate cancer asnon-metastatic or as lacking the propensity to metastasize. In specificembodiments, the markers used are OCT3/4, Nanog, and Sox2. In otherspecific embodiments, the markers used are OCT3/4, Nanog, Sox2, andc-Myc.

In still another aspect, the invention provides a method of determiningthe prognosis of a subject having neoplasia or prostate cancer, themethod comprising determining the level of a nucleic acid molecule orpolypeptide Marker selected from any one or more of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and uPAR in a biological sample derived from thesubject, relative to the level present in a reference. In oneembodiment, an increase in the level of each of said Markers identifiesor diagnoses the subject as having a poor prognosis. In anotherembodiment, an increase in the level of Sox2 identifies or diagnoses thesubject as having a poor prognosis. In yet another embodiment, theabsence of alteration in the level of one or more of said Markersidentifies or diagnoses the subject as having a good prognosis. In stillanother embodiment, the absence of alteration in the level of Sox2identifies or diagnoses the subject as having a good prognosis. Inspecific embodiments, the markers used are OCT3/4, Nanog, and Sox2. Inother specific embodiments, the markers used are OCT3/4, Nanog, Sox2,and c-Myc.

In another aspect, the invention provides a method of selecting anappropriate therapy for a subject having neoplasia or prostate cancer,the method comprising comparing the level of a Marker selected from anyone or more of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPARnucleic acid molecule or polypeptide in a biological sample derived fromthe subject, relative to the level present in a reference, wherein thean increase in the level of all of said Markers indicates thataggressive therapy is appropriate for the subject, and the absence of anincrease in the level of all of said Markers indicates that conventionaltherapy is appropriate. In specific embodiments, the markers used areOCT3/4, Nanog, and Sox2. In other specific embodiments, the markers usedare OCT3/4, Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a method of monitoringneoplasia or prostate cancer therapy in a subject, the method comprisingdetermining the level of a Marker selected from any one or more ofOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR nucleic acidmolecule or polypeptide in a biological sample derived from the subject,relative to the level present in a reference, wherein a reduction in thelevel of said marker. In specific embodiments, the markers used areOCT3/4, Nanog, and Sox2. In other specific embodiments, the markers usedare OCT3/4, Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a method of identifying ordiagnosing a neoplasia or prostate cancer as resistant to treatment witha conventional therapy, the method comprising identifying or detectingan increased level of a Marker selected from any one or more of OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR nucleic acid molecule orpolypeptide in a biological sample derived from the subject, relative tothe level present in a reference, wherein the increased level of saidMarkers identifies or diagnoses the neoplasia or prostate cancer asresistant to treatment with a conventional therapy. In specificembodiments, the markers used are OCT3/4, Nanog, and Sox2. In otherspecific embodiments, the markers used are OCT3/4, Nanog, Sox2, andc-Myc.

In another aspect, the invention provides a method of selecting atreatment for a subject diagnosed as having neoplasia or prostatecancer, the method involving quantifying the level of OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, and uPAR in a biologic sample from thesubject relative to a reference, wherein the presence or level ofexpression of OCT3/4, Nanog, Sox2, c-Myc or Klf4 is indicative of atreatment; and selecting a treatment. In specific embodiments, themarkers used are OCT3/4, Nanog, and Sox2. In other specific embodiments,the markers used are OCT3/4, Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a method of selecting atreatment for a subject diagnosed as having neoplasia or prostatecancer, the method involving quantifying the level of OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, and uPAR in a subject sample; andselecting a treatment for the subject, wherein the treatment is selectedfrom any one or more of surveillance, surgery, hormone therapy,chemotherapy, and radiotherapy. In specific embodiments, the markersused are OCT3/4, Nanog, and Sox2. In other specific embodiments, themarkers used are OCT3/4, Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a method for determining theMarker profile of a neoplasia or prostate cancer, the method comprisingquantifying the level of two or more Markers selected from any one ormore of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR in abiologic sample, wherein the level of Marker in the sample relative tothe level in a reference determines the Marker profile of the prostaticneoplasia. In specific embodiments, the markers used are OCT3/4, Nanog,and Sox2. In other specific embodiments, the markers used are OCT3/4,Nanog, Sox2, and c-Myc.

In another aspect, the invention provides a kit for the analysis ofOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR, the kitcomprising at least one polynucleotide or polypeptide capable ofspecifically binding or hybridizing to an OCT3/4, Nanog, Sox2, c-Myc,Klf4, Keratin 8, and uPAR polypeptide or nucleic acid molecule, anddirections for using the primer or antibody for the analysis of OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR. In one embodiment, thepolypeptide is an antibody detected by fluorescence, by autoradiography,by an immunoassay, by an enzymatic assay, or by a colorimetric assay. Inspecific embodiments, the markers used are OCT3/4, Nanog, and Sox2. Inother specific embodiments, the markers used are OCT3/4, Nanog, Sox2,and c-Myc. In various embodiments, the agents are primers (e.g., havingthe sequences shown in Table 2) or antibodies.

In another aspect, the invention provides a microarray comprising atleast two (e.g., 2, 3, 4, or 5) nucleic acid molecules, or fragmentsthereof, bound to a solid support, wherein the two nucleic acidmolecules are any one or more of OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, and uPAR.

In various embodiments of any of the above aspects, the method reducesor measures the expression of OCT3/4, Nanog, and Sox2, nucleic acidmolecules or polypeptides. In other various embodiments of any of theabove aspects, the method reduces or measure the expression of OCT3/4,Nanog, Sox2, and c-Myc nucleic acid molecules or polypeptides.

In various embodiments of any of the above aspects, the biologicalsample is a biologic fluid (e.g., blood, blood serum, plasma, saliva,urine, seminal fluids, and ejaculate) or tissue (e.g., prostate tissue).In specific embodiments, the biological sample is a blood sample (e.g.,peripheral blood). In specific embodiments, the biological or tissuesample contains peripheral blood mononuclear cells (PBMC). In variousembodiments of any of the above aspects, the level of Sox2 polypeptideor nucleic acid molecule is determined. In various embodiments of any ofthe above aspects, the reference is the level of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and uPAR polypeptide or nucleic acid moleculepresent in a control sample (e.g., a control sample derived from ahealthy subject or a subject with a non-metastatic prostate cancer or acontrol sample derived from the same subject at an earlier point intime). In specific embodiments, the reference is the level of Sox2polypeptide or nucleic acid molecule present in a control sample. Instill other embodiments of any of the above aspects, the method reducesor measures the expression of any two, three, four, or five of OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR nucleic acid molecules orpolypeptides.

In various embodiments of any of the above aspects, the method furthercomprises detecting the level of E-cadherin nucleic acid molecules orpolypeptides in a biological sample derived from the subject. In variousembodiments of any of the above aspects, the method further comprisesidentifying or detecting an increase in E-cadherin nucleic acidmolecules or polypeptides in a biological sample derived from thesubject, relative to the level present in a reference. In variousembodiments of any of the above aspects, the method further comprisesisolating or selecting cell in a biological sample derived from thesubject having an increase in E-cadherin nucleic acid molecules orpolypeptides, relative to the level present in a reference. In variousembodiments of any of the above aspects, isolating or selecting cellsthat bind an E-cadherin capture reagent from the biological sample priorto analysing said cells for a Marker nucleic acid molecule orpolypeptide. In various embodiments of any of the above aspects, thebiological sample comprises cells selected for E-cadherin expression. Invarious embodiments of any of the above aspects, E-cadherin is used toisolate or select a cell for E-cadherin expression. In variousembodiments of any of the above aspects, the cell binds a capturereagent (e.g., antibody, antibody fragment, or aptamer). In variousembodiments of any of the above aspects, the capture reagent is fixed toa substrate. In various embodiments of any of the above aspects, thecell is selected by flow cytometry.

The invention provides compositions and methods for diagnosing, treatingor preventing neoplasia (e.g., prostate cancer). Other features andadvantages of the invention will be apparent from the detaileddescription, and from the claims.

DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them below, unlessspecified otherwise.

By “OCT3/4 polypeptide” is meant a polypeptide or fragment thereofhaving at least about 85% amino acid identity to NCBI Accession No.NP_(—)002692 and having DNA binding activity.

By “OCT3/4 nucleic acid molecule” is meant a polynucleotide encoding anOCT3/4 polypeptide. An exemplary OCT3/4 nucleic acid molecule isprovided at NCBI Accession No. NM_(—)203289.

By “NANOG polypeptide” is meant a polypeptide or fragment thereof havingat least about 85% amino acid identity to NCBI Accession No.NP_(—)079141.2 and having DNA binding activity.

By “NANOG nucleic acid molecule” is meant a polynucleotide encoding aNANOG polypeptide. An exemplary NANOG nucleic acid molecule is providedat NCBI Accession No. NM_(—)024865.2.

By “SOX2 polypeptide” is meant a polypeptide or fragment thereof havingat least about 85% amino acid identity to NCBI Accession No.NP_(—)003097 and having DNA binding activity.

By “SOX2 nucleic acid molecule” is meant a polynucleotide encoding aSOX2 polypeptide. An exemplary SOX2 nucleic acid molecule sequence isprovided at NCBI Accession No. NM_(—)003106.

By “C-MYC polypeptide” is meant a polypeptide or fragment thereof havingat least about 85% amino acid identity to NCBI Accession No.NP_(—)002458.

By “C-MYC nucleic acid molecule” is meant a polynucleotide encoding aC-MYC polypeptide. An exemplary C-MYC nucleic acid molecule sequence isprovided at NCBI Accession No. NM_(—)002467.

By “KLF4 polypeptide” is meant a polypeptide or fragment thereof havingat least about 85% amino acid identity to NCBI Accession No.NP_(—)004226 and having DNA binding activity.

By “KLF4 nucleic acid molecule” is meant a polynucleotide encoding aKLF4 polypeptide. An exemplary KLF4 nucleic acid molecule sequence isprovided at NCBI Accession No. NM_(—)004235.

By “E-cadherin polypeptide” is meant a polypeptide or fragment thereofhaving at least about 85% amino acid identity to NCBI Accession No.CAA78353 and having cell surface expression.

By “E-cadherin nucleic acid molecule” is meant a polynucleotide encodingan E-cadherin polypeptide. An exemplary E-cadherin nucleic acid moleculesequence is provided at NCBI Accession No. NM_(—)004360.

By “keratin 8 polypeptide” is meant is meant a polypeptide or fragmentthereof having at least about 85% amino acid identity to NCBI AccessionNo. NP_(—)002264 and having biological activity.

By “keratin 8 polynucleotide” is meant a nucleic acid molecule encodinga keratin 8 polypeptide. An exemplary keratin 8 polynucleotide isprovided at NCBI Accession No. AF257789.

By “urokinase-type plasminogen activator receptor (uPar) polypeptide” ismeant a protein having at least about 85% amino acid identity to NCBIAccession No. AAF71751 that functions in regulation of cell-surfaceplasminogen activation.

By “uPar polynucleotide” is meant a nucleic acid molecule encoding auPar polypeptide. An exemplary uPar polynucleotide sequence is providedat NM_(—)002273.

Select exemplary sequences delineated herein are shown at FIG. 26.

By “alteration” is meant an increase or decrease. An alteration may beby as little as 1%, 2%, 3%, 4%, 5%, 10%, 20%, 30%, or by 40%, 50%, 60%,or even by as much as 75%, 80%, 90%, or 100%.

By “biologic sample” is meant any tissue, cell, fluid, or other materialderived from an organism.

By “cancer stem cell” or “stem-like cancer-initiating cell” is meantcells that can neoplastic and can undergo self-renewal as well asabnormal proliferation and differentiation. Functional features ofcancer stem cells are that they are tumorigenic; they can give rise toadditional neoplastic cells by self-renewal; and they can give rise tonon-tumorigenic neoplastic cells. Without being bound to any particulartheory, cancer stem cells contribute to the development of metastaticcancer.

By “clinical aggressiveness” is meant the severity of the neoplasia.Aggressive neoplasias are more likely to metastasize than lessaggressive neoplasias. While conservative methods of treatment areappropriate for less aggressive neoplasias, more aggressive neoplasiasrequire more aggressive therapeutic regimens.

By “inhibitory nucleic acid” is meant a double-stranded RNA, siRNA,shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof,that when administered to a mammalian cell results in a decrease (e.g.,by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a targetgene. Typically, a nucleic acid inhibitor comprises at least a portionof a target nucleic acid molecule, or an ortholog thereof, or comprisesat least a portion of the complementary strand of a target nucleic acidmolecule.

By “capture reagent” is meant a reagent that specifically binds anucleic acid molecule or polypeptide to select or isolate the nucleicacid molecule or polypeptide. Capture reagents may be used to select orisolate a cell expressing a polypeptide or Marker on the surface of thecell by specifically binding the cell surface expressed polypeptide orMarker (e.g., E-cadherin).

By “neoplasia” is meant any disease that is caused by or results ininappropriately high levels of cell division, inappropriately low levelsof apoptosis, or both. For example, cancer is an example of a neoplasia.Examples of cancers include, without limitation, prostate cancer,leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, acute myeloblastic leukemia, acute promyelocyticleukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acuteerythroleukemia, chronic leukemia, chronic myelocytic leukemia, chroniclymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease,non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chaindisease, and solid tumors such as sarcomas and carcinomas (e.g.,fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterinecancer, testicular cancer, lung carcinoma, small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma,meningioma, melanoma, neuroblastoma, and retinoblastoma).Lymphoproliferative disorders are also considered to be proliferativediseases.

By “reference” is meant a standard of comparison. For example, theOCT3/4, NANOG, SOX2, C-MYC or KLF4 polypeptide or polynucleotide levelpresent in a patient sample may be compared to the level of saidpolypeptide or polynucleotide present in a corresponding healthy cell ortissue or in a neoplastic cell or tissue that lacks a propensity tometastasize.

By “periodic” is meant at regular intervals. Periodic patient monitoringincludes, for example, a schedule of tests that are administered daily,bi-weekly, bi-monthly, monthly, bi-annually, or annually.

By “severity of neoplasia” is meant the degree of pathology. Theseverity of a neoplasia increases, for example, as the stage or grade ofthe neoplasia increases.

By “Marker profile” is meant a characterization of the expression orexpression level of two or more polypeptides or polynucleotides.

Nucleic acid molecules useful in the methods of the invention includeany nucleic acid molecule that encodes a polypeptide of the invention ora fragment thereof. Such nucleic acid molecules need not be 100%identical with an endogenous nucleic acid sequence, but will typicallyexhibit substantial identity. Polynucleotides having “substantialidentity” to an endogenous sequence are typically capable of hybridizingwith at least one strand of a double-stranded nucleic acid molecule. By“hybridize” is meant pair to form a double-stranded molecule betweencomplementary polynucleotide sequences (e.g., a gene described herein),or portions thereof, under various conditions of stringency. (See, e.g.,Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A.R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less thanabout 750 mM NaCl and 75 mM trisodium citrate, preferably less thanabout 500 mM NaCl and 50 mM trisodium citrate, and more preferably lessthan about 250 mM NaCl and 25 mM trisodium citrate. Low stringencyhybridization can be obtained in the absence of organic solvent, e.g.,formamide, while high stringency hybridization can be obtained in thepresence of at least about 35% formamide, and more preferably at leastabout 50% formamide. Stringent temperature conditions will ordinarilyinclude temperatures of at least about 30° C., more preferably of atleast about 37° C., and most preferably of at least about 42° C. Varyingadditional parameters, such as hybridization time, the concentration ofdetergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion orexclusion of carrier DNA, are well known to those skilled in the art.Various levels of stringency are accomplished by combining these variousconditions as needed. In a preferred: embodiment, hybridization willoccur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. Ina more preferred embodiment, hybridization will occur at 37° C. in 500mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100.mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferredembodiment, hybridization will occur at 42° C. C. in 250 mM NaCl, 25 mMtrisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Usefulvariations on these conditions will be readily apparent to those skilledin the art.

For most applications, washing steps that follow hybridization will alsovary in stringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25° C., more preferably of atleast about 42° C., and even more preferably of at least about 68° C. Ina preferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42.degree. C. in 15 mM NaCl, 1.5 mM trisodiumcitrate, and 0.1% SDS. In a more preferred embodiment, wash steps willoccur at 68° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.Additional variations on these conditions will be readily apparent tothose skilled in the art. Hybridization techniques are well known tothose skilled in the art and are described, for example, in Benton andDavis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad.Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in MolecularBiology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guideto Molecular Cloning Techniques, 1987, Academic Press, New York); andSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, New York.

By “substantially identical” is meant a polypeptide or nucleic acidmolecule exhibiting at least 50% identity to a reference amino acidsequence (for example, any one of the amino acid sequences describedherein) or nucleic acid sequence (for example, any one of the nucleicacid sequences described herein). Preferably, such a sequence is atleast 60%, more preferably 80% or 85%, and more preferably 90%, 95% oreven 99% identical at the amino acid level or nucleic acid to thesequence used for comparison.

Sequence identity is typically measured using sequence analysis software(for example, Sequence Analysis Software Package of the GeneticsComputer Group, University of Wisconsin Biotechnology Center, 1710University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, orPILEUP/PRETTYBOX programs). Such software matches identical or similarsequences by assigning degrees of homology to various substitutions,deletions, and/or other modifications. Conservative substitutionstypically include substitutions within the following groups: glycine,alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid,asparagine, glutamine; serine, threonine; lysine, arginine; andphenylalanine, tyrosine. In an exemplary approach to determining thedegree of identity, a BLAST program may be used, with a probabilityscore between e⁻³ and e⁻¹⁰⁰ indicating a closely related sequence.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show the identification of stem cells from metastaticprostate cancer (PCa) cell lines. FIG. 1A shows the results of RT-PCRanalysis for the detection of mRNA levels of CD133, Nanog and OCT3/4 inDU145, LNCaP and PC3 prostate cancer cell lines. Data were normalized toβ-actin expression. Representative results of three independentexperiments are shown. FIG. 1B shows Western blot analysis for thedetection of protein levels of Nanog and OCT3/4 in DU145, LNCaP and PC3PC cell lines. Data were normalized to β-actin expression.Representative results of three independent experiments are shown. FIG.1C shows DU145, LNCaP and PC3 cells immunostained for OCT3/4 (red),Nanog (green) and the merge of OCT3/4 and Nanog (OCT3/4+Nanog, yellow).Phase contrast images served as controls. Representative results ofthree independent experiments are shown.

FIGS. 2A-2C show the identification of stem cell-like tumor cells withpluripotent stem cell reprogramming factors in prostate cancer celllines. FIG. 2A shows the results of RT-PCR analysis for detectingexpression levels of OCT3/4, SOX2, Nanog, c-Myc and Klf4 in DU145 andPC3 cell lines, with human Embryonic Stem Cells (hESC) as the control.Data were normalized to β-actin expression. Representative results ofthree independent experiments are shown. FIG. 2B shows the results ofWestern blot analysis for detecting expression levels of OCT3/4, SOX2,Nanog, c-Myc and Klf4 in DU145 and PC3 cell lines, with human EmbryonicStem Cells (hESC) as the control. Data were normalized to β-actinexpression. Representative results of three independent experiments areshown. FIG. 2C depicts, images of DU145 and PC3 cells immunostained forOCT3/4 (red), SOX2 (green), DAPI (blue); OCT3/4 and SOX2 staining werealso merged. Magnification is 40× and the scale bar represents 20 μm.Representative results of three independent experiments are shown.

FIGS. 3A-3D show that prostate cancer stem cells express E-cadherin andcan be sorted from non-stem prostate cancer cells by FACS sorting usingE-cadherin. FIG. 3A shows the identification of surface markers forisolating tumor-initiating cells (cancer stem cells, CSC) frommetastatic prostate cell lines. DU145, LNCaP and PC3 cells wereimmuno-stained for OCT3/4 (red), E-cadherin (green), DAPI (blue) and themerge of all pictures. Representative results of three independentexperiments are shown. FIG. 3B depicts the sorting and analysis ofprostate cancer stem cells and non-stem prostate cancer cells from theDU145 cell line by flowcytometry. Shown are the E-cadherin expressionbefore (left) and after sorting of high expression (right, top) and lowexpression (right, bottom) tumor cells; the value in each graphrepresents the percentage of enriched population; dead cells were gatedby propidium iodide (PI). Representative results of three experimentsare shown. FIG. 3C depicts fluorescent-activated cell sorting (FACS) ofstem cells sorted from DU145, LNCaP and PC3 prostate cell lines usingE-cadherin as a marker. FIG. 3D shows RT-PCR analysis for the detectionof mRNA levels of E-cadherin, Nanog and OCT3/4 in DU145, LNCaP and PC3prostate cancer cell lines. Data were normalized to β-actin expression.Representative results of three independent experiments are shown.

FIGS. 4A-4F depict the isolation of stem cell-like prostate tumor cellsby FACS sorting using E-cadherin. FIG. 4A shows the screening andidentification of surface markers for isolating stem cell-like cellsfrom prostate cell lines. DU145 and PC3 cells were immunostained forOCT3/4 (red), E-cadherin (green), DAPI (blue); OCT3/4 and E-cadherin(E-cad) staining were also merged. Magnification is 40× and the scalebar is 20 μm. FIGS. 4B and 4C are graphs depicting the phenotypicanalysis of DU145 and PC3 cells using double-staining with E-cadherinand CD44 (FIG. 4B) or Integrin-α2β1 (FIG. 4C). Cells were gated on theE-cadherin+ (green) or E-cadherin− (blue) population. FIG. 4D is a graphdepicting flow cytometry analysis of DU145 and PC3 cells showingE-cadherin expression. FIG. 4E is a graph depicting isotype matchedcontrols of flow cytometry analysis of DU145 and PC3 used to setanalysis gates for E-cadherin cell sorting. FIG. 4F shows RT-PCRanalysis detecting expression levels of OCT3/4, SOX2, Nanog, c-Myc andKlf4 in E-cadherin⁺ and E-cadherin⁻ cells isolated from DU145 and PC3cells. Data were normalized to β-actin expression. Representativeresults of three independent experiments are shown.

FIGS. 5A-5D show that prostate cancer stem cells isolated frommetastatic prostate cancer cell lines are clonigenic, proliferative, candifferentiate, and are invasive. FIG. 5A shows the clonigenic properitesof prostate tumor stem cells (CSC) and non-stem prostate cancer cells(Non-CSC) in a colony forming assay. E-cadherin⁺ and E-cadherin⁻ cellsisolated from metastatic prostate cancer cell lines by FACS analysiswere cultured in semisolid medium of soft agar for 2-3 weeks untilcolonies were well-formed. The colonies were counted to determine thenumber of clones. Data represent the mean±SD from two independentexperiments. **p<0.01. Representative plates from each group are shownin the insets above. FIG. 5B depicts representative images of spheroidculture assay using E-cadherin sorted cells. Western blot comparingunsorted parental line (P) to E-cadherin⁺ spheroids (S) showing proteinlevels of OCT3/4, SOX2, and E-cadherin. Data were normalized to β-actinexpression. Magnification is 5×. FIG. 5C shows representative images ofsorted prostate tumor stem cells (CSC) and non-stem prostate cancercells (Non-CSC) on plates after 3 days culture. Representative phaseimages are on the left panels; representative immunofluorescence imagesdetecting E-Cadherin are on the center panels and β-catenin on the rightpanels. Magnification is 5× for phase contrast and 40× forimmunofluorescence. The scale bar is 100 μm for phase contrast and 20 μmfor immunofluorescence. FIG. 5D shows representative images of atranswell migration assay demonstrating the invasiveness of prostatetumor stem cells (CSC) and non-stem prostate cancer cells (Non-CSC).Representative phase images at 10× magnification are on the top panels;representative phase images at 20× magnification are on the bottompanels.

FIGS. 6A-6C show that prostate cancer stem cells isolated frommetastatic stem cell lines are tumorigenic in SCID mice. FIG. 6A showsphotographs of xenograft tumors in mice (five mice per group) injectedwith prostate tumor stem cells (CSC) and non-stem prostate cancer cells(Non-CSC). FIG. 6B is a graph depicting the tumorigenic potential ofisolated tumor-initiating cells from the PC3 prostate cancer cell linein SCID mice after subcutaneous injection (sorted E-cadherin⁺, bluediamond; E-cadherin⁻, pink square). Data concerning tumor volume aremean±SD from five mice in each group. Representative results of twoexperiments are shown. FIG. 6C is a graph depicting the tumorigenicpotential of isolated tumor-initiating cells from the DU145 prostatecancer cell line in SCID mice after subcutaneous injection. Dataconcerning tumor volume are mean±SD from five mice in each group.Representative results of two experiments are shown.

FIGS. 7A and 7B show the expression of pluripotent stem cell genes inmetastatic prostate tumor-initiating stem cells. FIG. 7A shows RT-PCRanalysis for the detection of mRNA levels of c-Myc, Klf4, OCT3/4 andSox2 in tumor-initiating cells (CSC) or non-tumor-initiating cells (NC)isolated from DU145, LNCaP and PC3 cells. Data were normalized toβ-actin expression. Representative results of three independentexperiments are shown. FIG. 7B shows Western-blot analysis for detectingprotein levels of c-Myc, Klf4, Nanog, OCT3/4 and Sox2 intumor-initiating cells isolated from DU145, LNCaP and PC3 cells. Datawere normalized to β-actin expression. Representative results of threeindependent experiments are shown.

FIGS. 8A-8D show that prostate cancer stem cells are present in humanprostate tumor tissue. FIG. 8A depicts a hypothetical model for theorigin and differentiation of cancer stem cells in prostate. FIG. 8Bshows RT-PCR analysis for the detection of mRNA levels of OCT3/4, Sox2,c-Myc, Nanog, prostate specific antigen (PSA) and androgen receptor (AR)in 4 independent samples of tumor tissue from primary human prostatecancer (PCa#1, PCa#2, PCa#3, PCa#4), isolated prostate cancer stem cells(Pca SC), embryonic stem cells (ESC) or dendritic cells (DC). Data werenormalized to β-actin expression. Representative results of threeindependent experiments are shown. FIG. 8C shows the expression of OCT3/4 (top panels) and SOX2 (lower panels) in human tissue samplesvisualized by immunohistochemical staining using antibodies specific forOCT 3/4 and SOX2 respectively. Representative images of normal prostate(left panels) and fetal testes (right panels) are shown. Images werecaptured with Zeiss Axioplan 2 upright microscope. Brown color stainedcells represent the positive cells. FIG. 8D shows the expression of OCT3/4 and SOX2 in human prostate tumor tissue. Representative images ofprostate tumor tissue visualized by Hematoxylin and Eosin (H&E) staining(upper left panel), immunohistochemical staining with IgG controantibodies (lower left panel), immunohistochemical staining with OCT 3/4antibodies (upper right panel, magnification in inset), andimmunohistochemical staining with SOX2 antibodies (lower right panel,magnification in inset). Images were captured with Zeiss Axioplan 2upright microscope. Brown color stained cells represent the positivecells.

FIGS. 9A-9F depict expression of pluripotent stem cell genes c-Myc,Klf4, Nanog, OCT3/4 and Sox2 in prostate cancer tissues. FIGS. 9A-9E aregraphs showing semiquantitative RT-PCR of OCT3/4 (FIG. 9A), Sox2 (FIG.9B), Nanog (FIG. 9C), c-Myc (FIG. 9D), and Klf4 (FIG. 9E) usingcommerically available prostate tissue panels (Origene TissueScan).Normal prostate (N, black), prostate tumor sphere cells (PS, crosshatch)and hESC (ES, gray) served as controls. Band intensities were calculatedusing AlphaEase software (AlphaInnotech). Transcript levels for eachcase were normalized to β-actin expression and are represented asrelative units standardized to the normal tissue pool. Representativeresults of three independent experiments are shown. Statisticalsignificance was set at p<0.05; * is statistically different from thenormal tissue pool and ‡ is statistically different from hESC. FIG. 9Fis a graph depicting the correlation of mRNA expression levels betweenSOX2 and OCT3/4 in tissue samples. The relative level of SOX2 expressionwas plotted against the relative level of OCT3/4 expression using thenormal tissue pool as reference and gave a Spearman correlationcoefficient of 0.4730 (p<0.0001).

FIGS. 10A-10C depict the immunohistochemical detection of OCT3/4 andSOX2 in human prostate cancer tissues. FIG. 10A provides images ofimmunostaining for OCT3/4 and SOX2 using prostate tissue arrays.Representative images from negative, low (<5%), intermediate (5-25%) andhigh (26-50%) percentage staining are shown. Brown color indicatespositive nuclear staining. Magnification is 20× with the inset at 40×and the scale bar is 70 μm. Representative results of at least twoindependent experiments are shown. FIGS. 10B and 10C are graphsclassifying different Gleason Score samples based on category ofstaining intensity for OCT3/4 (FIG. 10B) and SOX2 (FIG. 10C). The redline represents the mean.

FIGS. 11A and 11B show that prostate cancer stem cells are resistant toirradiation. FIG. 11A shows Western blot analysis performed on thesamples of prostate cancer stem cells using antibodies to Sox2, Oct3/4,Nanog, E-cadherin, β-Catenin and Actin in which the prostate cancercells were exposed to various doses of radiation, including 0 Gy, 2 Gy,4 Gy, 6 Gy, and 8 Gy doses. FIG. 11B is a graph depicting the survivingfraction of prostate cancer stem cells (CSC) and non-stem prostatecancer cells (Non-CSC) from the samples exposed to radiation (0 Gy, 2Gy, 4 Gy, 6 Gy, 8 Gy and 10 Gy).

FIGS. 12A and 12B show that prostate cancer stem cells are resistant toDocetaxel. FIG. 12A shows Western blot analysis performed on samples ofprostate cancer stem cells using antibodies to Sox2, Oct3/4, Nanog,E-cadherin, β-Catenin and Actin in which the. prostate cancer cells wereexposed to various doses of docetaxel, including 1 nM, 2 nM, 5 nM, and10 nM doses. FIG. 12B is a graph depicting the cell viability ofprostate cancer stem cells (CSC) and non-stem prostate cancer cells(Non-CSC) observed for up to 72 hours after treatment with Docetaxel.Cell viability was determined by quantifying the surviving prostatecancer stem cells (CSC) and non-stem prostate cancer cells (Non-CSC) inthe samples exposed to 5 nM Docetaxel at Day 0, 1, 2 and 3.

FIGS. 13A-13C show that prostate cancer stem cells are immune privilegedor immunosuppressive. FIG. 13A shows RT-PCR analysis for the detectionof mRNA levels of LMP2, LMP7, TAP1, TAP2, and Tapasin intumor-initiating stem cells (Ecad+) or non-tumor-initiating cells(Ecad−) isolated from DU145, LNCaP and PC3 cells. Data were normalizedto β-actin expression. Representative results of three independentexperiments are shown. FIG. 13B shows RT-PCR analysis for the detectionof mRNA levels of CD44, Ecad, Nanog, OCT3/4, and TERT intumor-initiating stem cells (CSC) or non-tumor-initiating cells(Non-CSC) isolated from LNCaP cells, which were used in the experimentin FIG. 13C. Data were normalized to β-actin expression. Representativeresults of three independent experiments are shown. FIG. 13C is a graphdepicting the data from Interferon-γ enzyme linked immunosorbent spot(IFN-γ ELISPOT) assays performed on prostate cancer stem cells (CSC) anddendritic cells (DC). Prostate cancer stem cells were untreated (CSC),treated with isotype-specific antibody (CSC+Iso Ab.), treated withantibody to E-cadherin (CSC+E-cad blocking), or treated with antibody toHLA-class I (CSC+HLA Blocking). Untreated dendritic cells were used as anegative control and dendritic cells expressing hTERT (hTERT DC) wereused as a positive control. Antigen-specific T-cells were mixed with thecells in the samples, plated, and the numbers spot forming coloniesquantified for each sample.

FIGS. 14A-14B show that siRNAs to transciption factors in prostate stemcells increase cell death in prostate stem cells. FIG. 14A shows RT-PCRanalysis examining the efficiency of siRNAs targeted against c-Myc,Klf4, Nanog, OCT3/4 and Sox2 in silencing mRNA expression ofcorresponding genes in prostate cancer stem cells. The mRNA levels ofthese genes in cells treated with control siRNA (Cntl-siR) were used ascontrols. Data were normalized to β-actin expression. Representativeresults of two independent experiments are shown. FIG. 14B shows flowcytometry analysis of CSC cells and Non-CSC cells from DU145, LNCaP andPC3 cells treated with siRNAs against c-Myc, Klf4, Nanog, OCT3/4 andSox2 for 24 hours. Cells were recovered and apoptotic cells weredetected using the annexin V and PI binding assay. Value in lower leftcorner represents the percentage of viable cells. Cells treated withcontrol siRNA were used as controls. Representative results of threeindependent experiments are shown.

FIGS. 15A-15D show that shRNAs or siRNAs that reduce the expression ofcertain transciption factors in prostate stem cells also decrease thetumorigenicity of the prostate stem cells. FIG. 15A provides a Westernblot showing decreased OCT3/4 or SOX2 protein levels in human DU145prostate cancer cells transfected with shRNA compared to thosetransfected with control shRNA. FIG. 15B is a graph depicting thetumorigenic potential of isolated tumor-initiating stem cells from theDU145 cell line is decreased when DU145 prostate cancer stem cells arepre-treated with Sox2 or Oct 3/4 shRNA in SCID mice. Tumors were notdetected in mice injected with stem cells pre-treated with Sox2 or Oct3/4 shRNA just under 70 days after injection. Unsorted DU145 cells(1×10⁵) were subcutaneously injected in SCID mice after treatment withOCT 3/4 (pink square), SOX2 (green diamond), or control shRNA (bluetriangle). Tumor volume data are reported as the mean±SD from the fourmice that developed tumors. Representative results of two independentexperiments are shown. FIG. 15C depicts representative images showingtumor development. The scale bar is 1 cm. FIG. 15D is a graph depictingthe tumorigenic potential of isolated tumor-initiating stem cells fromthe DU145 cell line is decreased when DU145 prostate cancer stem cellsare pre-treated with Sox2 or Oct 3/4 siRNA in SCID mice. Data concerningtumor volume are mean±SD from five mice in each group.

FIGS. 16A-C show the detection of pluripotent stem cell reprogrammingfactors in the peripheral blood of patients with prostate cancer. FIG.16A shows that increased levels of OCT3/4, SOX2, and Nanog were detectedin peripheral blood samples of prostate cancer patients compared tosamples from healthy donors. RT-PCR analysis was used to detect mRNAlevels of OCT3/4, Sox2, Nanog, and β-microglobulinin samples from humanembryonic stem cells (ES), normal peripheral blood mononuclear cellspooled from a minimum of 10 donors (N), healthy individuals (N1-N3), andprostate cancer patients (P1-P9). Human embryonic stem cells were usedas positive control and β-microglobulin was used as an internal control.FIG. 16B shows that increased levels of OCT3/4, SOX2, and Nanog weredetected in peripheral blood samples of prostate cancer patientscompared to a sample from normal peripheral blood mononuclear cells.RT-PCR analysis was used to detect mRNA levels of OCT3/4, Sox2, Nanog,c-Myc, Klf4, Keratin 8, uPAR, and β-microglobulin in samples from humanembryonic stem cells (ES), normal peripheral blood mononuclear cellspooled from a minimum of 10 donors (N), and prostate cancer patients(P1-P9). Human embryonic stem cells were used as positive control andβ-microglobulin was used as an internal control. FIG. 16C is a graphshowing that significantly increased levels of OCT3/4, SOX2, and Nanogwere detected in peripheral blood mononuclear cells (PBMC) of prostatecancer patients compared to normal peripheral blood mononuclear cells.Semi-quantitative RT-PCR analysis was applied to PBMC from 9 prostatecancer patients and compared to a pooled normal PBMC from 13 normalhealthy donors (10 males and additional 3 individual normal healthydonors). Band intensities were calculated using commercially availablequantitation software (AlphaEase software, AlphaInnotech). Transcriptlevels for each case were normalized to β-microglobulin expression andare represented in the graph as relative units standardized to theaveraged normal expression in PBMC. Samples are from normal peripheralblood mononuclear cells pooled from a minimum of 10 donors (N), and PBMCfrom prostate cancer patients (P1-P9). Arrows indicate patient status:alive without disease (AWD); dead of disease (DOD).

FIG. 17 shows levels of pluripotent stem cell reprogramming factorsOCT3/4, Sox2, Nanog, c-Myc, Klf4, Keratin 8, uPAR, and β-microglobulinin peripheral blood mononuclear cells (PBMC) samples of metastaticprostate cancer patients undergoing vaccination for human telomerasereverse transcriptase (hTERT) or lysosome-associated membrane protein-1(LAMP) hTERT. RT-PCR analysis was used to assess the expression level ofOCT3/4, SOX2, Nanog, c-Myc, and Klf4 in PBMC of prostate cancerpatients. β-microglobulin levels was used as internal control. Patientsin the study were immunized with six weekly doses of hTERT(16-TERT), sixweekly doses of LAMP hTERT(14-LAMP; 19-LAMP), or three weekly cell dosesof hTERT-(4-TERT; 9-TERT; 11-TERT).

FIGS. 18A-18E show that DU145 and PC3 prostate cancer cells isolated asE-cad⁺ are invasive. FIGS. 18A and 18B show that E-cad+ and E-cad⁻ cellswere isolated by FACS sorting of DU145 and PC3 cells, respectively.Parental DU145 or PC3 cells were trypsinized, incubated for 10 hr torecover the adhesion molecules in 10% FBS containing medium, and stainedwith a PE-conjugated E-cadherin antibody and analyzed by flow cytometry.FIG. 18C shows Western blotting analysis for E-cadherin, OCT3/4, SOX2,Nanog and Klf4 in parental cells and spheroids formed by E-cad⁺ DU145and PC3 cells. FIG. 18D depicts representative images of invaded E-cad⁺and E-cad⁻ DU145 and PC3 cells. E-cad⁺ and E-cad⁻ sorted subpopulationswere plated in invasion chambers immediately after cell sorting.Twenty-four hours later non-invaded (top-chamber) cells were removed,and the invaded cells were stained with crystal violet. FIG. 18E showsthat invaded DU145 and PC3 cells express E-cadherin. Top-chamber (a andc) or invaded (b and d) parental DU145 and PC3 cells, respectively, wereimmunostained with an E-cadherin antibody at the conclusion of a 24 hrinvasion assay.

FIGS. 19A-19J show that E-cad⁺ subpopulations isolated from DU145 andPC3 prostate cancer cells have a high invasive capacity. FIG. 19A is agraph depicting number of invaded cells in an assay using E-cad⁺ andE-cad⁻ subpopulations sorted from DU145 and PC3 cells. FIG. 19B is agraph depicting the growth in culture of invaded E-cad⁺ and E-cad⁻ DU145cells. After a 24 h invasion period, invaded E-cad⁺ and E-cad⁻ cellswere plated in adherent culture conditions for 3 days and then counted.FIG. 19C depicts phase-contrast images of invaded E-cad⁺ DU145 and PC3cells after 3 days of culture. Invaded E-cad⁺ cells exhibited aholoclone-type colony formation (top panels, ×10) and E-cadherinexpression (labeled green, bottom panels, ×40). FIG. 19D is a graphdepicting the in vitro quantification of prostate cell spheroids formedby invaded E-cad⁺ cells. The data are expressed as the percentage ofspheroids formed per 500 seeded cells ±SD. FIG. 19E depictsphase-contrast images of DU145 spheroids seeded by invaded E-cad⁺ (panela) and E-cad⁻ (panel b) cells (×10), and images of spheroidsimmunostained with antibodies against E-cad (green; panel c) and CD44(red; panel d) (×40). FIG. 19F depicts phase-contrast images of PC3spheroids from invaded E-cad⁺ (panel a) and E-cad⁻ (panel b) cells(×10), and images of spheroids immunostained with antibodies againstE-cad (green; panel c) and CD44 (red; panel d) (×40). FIG. 19G depictsimmunofluorescence images of both non-invaded (top; panels a and b) andinvaded (bottom; panels c and d) cells from plated E-cad⁺ and E-cad⁻subpopulations of DU145 cells stained with E-cadherin. Magnification,×40. FIG. 19H depicts immunofluorescence images of both non-invaded(top, panels a and b) and invaded (bottom, panels c and d) cells fromplated E-cad⁺ and E-cad⁻ subpopulations of PC3 cells stained withE-cadherin. Magnification, ×40. FIG. 19I depicts immunofluorescenceimages of E-cad⁺ cells (DU145, panel a; PC3, panel b) in the top chamberafter 1 hr in the invasive assay stained for E-cadherin (×10), FIG. 19Jdepicts immunofluorescence images of invaded E-cad⁺ cells (DU145, panela; PC3, panel b) at the bottom of the membrane after 4 hr in theinvasive assay stained for E-cadherin (×40).

FIGS. 20A-20D show that E-cad⁺ DU145 and PC3 cells modulate E-cadherinexpression during invasion. FIGS. 20A and 20B depict immunofluorescenceimages of invaded E-cad⁺ cells immunostained for E-cadherin. After 4 hof culture (t=0), top-chamber (non-invaded) cells were removed, and theinvasion chambers were incubated for an additional 5, 10, or 15 h. Ateach time point, DU145 (FIG. 20A) and PC3 (FIG. 20B) cells wereimmunostained for E-cadherin. Magnification, ×40. FIGS. 20C and 20Ddepict graphs showing the relative E-cadherin and Slug expression levelsin E-cad⁺ cells from DU145 (FIG. 20C) and PC3 (FIG. 20D) cells in thetop chambers were evaluated by qPCR at various times (0, 2, 4, 8, 16 and24 h). Expression levels were normalized to actin. The data are reportedas the mean±SEM.

FIGS. 21A-21I show that OCT3/4 and SOX2 are required for prostate cancercell invasion. FIG. 21A shows Western blot analysis for the expressionlevels of E-cadherin, SOX2, OCT3/4, β-catenin, c-Myc, c-Met, Nestin, andtubulin (loading control) in DU145 and PC3 cells E-cad knockdowncompared to control cells. (C-control lane; E-cad-E-cad siRNA lane).FIGS. 20B and 20C are graphs showing the number of DU145 (FIG. 20B) andPC3 (FIG. 20C) cells invading through Matrigel-coated membranes aftertransient transfection with E-cadherin or control siRNA. All chamberassays were performed in triplicate (See also FIG. 22A). FIG. 21D showsWestern blot analysis of E-cadherin, SOX2, OCT3/4, β-catenin, c-Myc,c-Met and Nestin in DU145 and PC3 SOX2 knockdown compared to controlcells. Actin was used as a loading control. (C-control lane; SOX2-SOX2siRNA lane). FIGS. 20E and 20F are graphs showing the invasion of DU145(FIG. 20E) and PC3 (FIG. 20F) SOX2 knockdown compared to control cells.FIG. 20G shows Western blot analysis of E-cadherin, SOX2, OCT3/4,β-catenin, c-Myc, c-Met and Nestin in DU145 and PC3 OCT3/4 knockdowncompared to control cells. Actin was used as a loading control.(C-control lane; OCT3/4-OCT3/4 siRNA lane). FIGS. 20H and 20I are graphsshowing the invasion of OCT3/4 knockdown DU145 (FIG. 20H) and PC3 (FIG.20I) compared to control cells.

FIGS. 22A-22C show the effects of E-cadherin, SOX2 or OCT3/4 knockdownin invasion of DU145 and PC3. FIG. 22A depicts representative images ofinvasion across a Matrigel-coated membrane for control and E-cadherinsiRNA-transfected DU145 and PC3 cells. Control and E-cadherin knockdowncells were plated in 500 ul serum free medium in the top chamber, and750 gl of 10% FBS-containing medium was used as the attractant in the 24well plates. After 24 h, cells remaining in the top-chamber cells wereremoved with cotton swabs, and invaded cells in the bottom chamber werestained with crystal violet. FIG. 22B depicts representative images ofinvasion across a Matrigel-coated membrane for control and SOX2shRNA-transfected DU145 and PC3 cells. Control and SOX2shRNA-transfected DU145 and PC3 cells were plated and treated asdescribed for FIG. 22A above. FIG. 22C depicts representative images ofinvasion across a Matrigel-coated membrane for control and OCT3/4shRNA-transfected DU145 and PC3 cells. Control and OCT3/4 shRNAtransfected DU145 and PC3 cells were plated and treated as described forFIG. 22A above.

FIG. 23 shows the effect of trypsin treatment on DU145 and PC3 cells.Expression of E-cadherin in DU145 and PC3 cells wasa analyzed by Westernblot. Tubulin was used as a loading control. Cells were directlyharvested with lysis buffer (L) or were collected by trypsinization (T).

FIGS. 24A and 24B depict a model of a role for E-cadherin modulation inprostate cancer cell invasion. FIG. 24A depicts proposed roles ofE-cadherin and EMT genes in stages progressive tumor formation (EMT andtransformation) and the development of the aggressive and franklymetastatic phenotype. FIG. 24B depicts a schema of the modulation ofE-cadherin expression in the post-epithelial to mesenchymal (post-EMT)acquisition of the aggressive phenotype. E-cadherin is indicative of thepre-EMT state. Parental DU145 and PC3 cells exhibiting a mixed-markerEMT phenotype are believed to have already undergone EMT and expressE-cadherin at relatively low levels. Highly metastatic lesions arisingfrom primary prostate tumors, but not the primary tumors themselves,exhibit high levels of E-cadherin. A proposed mechanism for post-EMTupregulation of E-cadherin is that plasticity of E-cadherin expressionis a permissive factor for cellular invasion, but requires expression ofthe embryonic stem cell markers SOX2 and OCT3/4.

FIGS. 25A-25B show that expression level of EMT related genes isdecreased in E-cad+ compared to E-cad− cells. FIG. 25A shows RT-PCRanalysis of Slug, Snail and Vimentin detected in parental DU145 and PC3cells. GADPH was used as an internal control. FIGS. 25B and 25C aregraphs showing quantitative PCR analysis of expression levels of mRNAsencoding E-cadherin, Slug, Snail and Vimentin in E-cad− relative toE-cad⁺ DU145 and PC3 cells, respectively. Gene expression levels werenormalized to actin. The data are reported as mean±SEM.

FIG. 26 provides exemplary sequences of human OCT3/4, Nanog, Sox2, c-Mycand Klf4 polypeptides and nucleic acid molecules.

DETAILED DESCRIPTION OF THE INVENTION

The invention features compositions and methods that are useful for thediagnosis, treatment and prevention of neoplasias (e.g., prostatecarcinoma), as well as for characterizing a neoplasia (e.g., prostatecarcinoma) to determine subject prognosis and aid in treatmentselection. The invention further provides compositions and methods formonitoring a patient identified as having a neoplasia (e.g., prostatecarcinoma). The present invention is based, at least in part, on thediscovery that pluripotent stem cell transcription factors, OCT3/4,Nanog, Sox2, c-Myc and Klf4, are expressed by prostate tumor-initiatingcells; that levels of OCT3/4, Sox2, Nanog, c-Myc, Klf4, Keratin 8, anduPAR are increased in the blood of subject identified as having prostatecarcinoma, that increases in Sox2 expression levels correlate with areduction in subject survival; and that Sox2 alone or in combinationwith Oct3/4 and/or Nanog and/or c-Myc may be used to characterize theprostate carcinoma to inform treatment selection and subject prognosis.In other embodiments, Sox2, Oct3/4, Nanog, and c-Myc are characterizedto inform treatment selection and subject prognosis. As reported in moredetail below, stem-like tumor-initiating cells were identified andisolated from primary prostate tumor tissue and three metastaticprostate tumor lines, and these cells exhibited a clear stem celltranscriptional signature. This discrete population of stem-liketumor-initiating cells possessed strong tumorgenicity andtransplantability in SCID mice and are resistant to the radiationtherapy and chemo-therapy. Furthermore, inhibition of any one of thesegenes in these cells resulted in significant apoptosis and necrosis.Prostate tumor-initiating cells may achieve pluripotency byreprogramming and expressing the combination of markers OCT3/4, Nanog,Sox2, c-Myc and Klf4 stem cell transcription factors. Importantly,increased expression of these markers, as well as Keratin 8, and uPAR isdetected in the peripheral blood of subjects identified as havingprostate carcinoma, and increased expression of Sox2, alone or incombination with increased expression of Oct3/4 and Nanog, has beenfound to correlate with reduced subject survival. Accordingly, theinvention provides diagnostic compositions that are useful inidentifying subjects as having or having a propensity to develop aprostate carcinoma, as well as methods of using these compositions toidentify a subject's prognosis, select a treatment regimen, and monitorthe subject before, during or after treatment.

In specific embodiments, the invention provides compositions and methodsfor characterizing the molecular profile of a neoplasia (e.g., prostatecancer) to identify those neoplasias that require immediate and/oraggressive therapeutic intervention from those neoplasias that could bemonitored for a period of weeks, months or years. In one embodiment, aprostate cancer that expresses low or undetectable levels of Sox2,Oct3/4, Nanog, and/or c-Myc is identified as a neoplasia that could bemonitored prior to therapeutic intervention. In another embodiment, aprostate cancer that expresses increased levels of one or all of Sox2,Oct3/4, Nanog, and/or c-Myc, relative to a normal cell or anon-aggressive prostate cancer cell, is identified as a neoplasia inneed of therapeutic intervention, immediate therapeutic intervention,and/or aggressive therapeutic intervention.

Diagnostics

The present invention features diagnostic assays for the detection ofneoplasias, benign prostatic hyperplasia, prostate cancer or thepropensity to develop such conditions. In one embodiment, levels of anyone or more of the following markers OCT3/4, SOX2, Nanog, c-Myc, Klf4,keratin 8, and uPAR are measured in a subject sample and used tocharacterize neoplasia, benign prostatic hyperplasia, prostate cancer orthe propensity to develop such conditions. In other embodiments, levelsof OCT3/4, SOX2, and/or Nanog, are characterized in a subject sample. Insome embodiments, levels of OCT3/4, SOX2, and Nanog are characterized ina subject sample. In other embodiments, levels of OCT3/4, SOX2, Nanog,and c-Myc are characterized in a subject sample. In other embodiments,levels of SOX2 are characterized, alone, or in combination with Oct3/4and/or Nanog. Standard methods may be used to measure levels of a markerin any biological sample.

Biological samples include tissue samples (e.g., cell samples, biopsysamples) and bodily fluids, including, but not limited to, blood, bloodserum, plasma, saliva, urine, seminal fluids, and ejaculate. Methods formeasuring levels of polypeptide include immunoassay, ELISA, westernblotting and radioimmunoassay. Elevated levels of SOX2 alone or incombination with one or more additional markers are considered apositive indicator of prostate cancer. The increase in SOX2, OCT3/4and/or Nanog levels may be by at least about 10%, 25%, 50%, 75% or more.The increase in SOX2, OCT3/4, and Nanog levels may be by at least about10%, 25%, 50%, 75% or more. The increase in SOX2, OCT3/4, Nanog, andc-Myc levels may be by at least about 10%, 25%, 50%, 75% or more. In oneembodiment, any increase in a marker of the invention is indicative ofprostate carcinoma. In one embodiment, an increase in SOX2 relative tonormal levels is indicative of prostate carcinoma. In anotherembodiment, levels of OCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, anduPAR are used to distinguish prostate carcinoma from benign prostatichyperplasia. In another embodiment, levels of OCT3/4, SOX2, and Nanogare used to distinguish prostate carcinoma from benign prostatichyperplasia. In another embodiment, levels of OCT3/4, SOX2, Nanog, andc-Myc are used to distinguish prostate carcinoma from benign prostatichyperplasia. In general, an increase in SOX2 polypeptide orpolynucleotide levels is indicative of prostate carcinoma or thepropensity to develop prostate carcinoma. If desired, cells present in abiologic sample derived from a subject are selected using a capturereagent that specifically binds E-cadherin prior to assaying the cellsfor the expression of a marker of the invention.

Any suitable method can be used to detect one or more of the markersdescribed herein. Successful practice of the invention can be achievedwith one or a combination of methods that can detect and, preferably,quantify the markers. These methods include, without limitation,hybridization-based methods, including those employed in biochip arrays,mass spectrometry (e.g., laser desorption/ionization mass spectrometry),fluorescence (e.g. sandwich immunoassay), surface plasmon resonance,ellipsometry and atomic force microscopy. Expression levels of markers(e.g., polynucleotides or polypeptides) are compared by procedures wellknown in the art, such as RT-PCR, Northern blotting, Western blotting,flow cytometry, immunocytochemistry, binding to magnetic and/orantibody-coated beads, in situ hybridization, fluorescence in situhybridization (FISH), flow chamber adhesion assay, ELISA, microarrayanalysis, or colorimetric assays. Methods may further include, one ormore of electrospray ionization mass spectrometry (ESI-MS), ESI-MS/MS,ESI-MS/(MS)^(n), matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF-MS), surface-enhanced laserdesorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS),desorption/ionization on silicon (DIOS), secondary ion mass spectrometry(SIMS), quadrupole time-of-flight (Q-TOF), atmospheric pressure chemicalionization mass spectrometry (APCI-MS), APCI-MS/MS, APCI-(MS)^(n),atmospheric pressure photoionization mass spectrometry (APPI-MS),APPI-MS/MS, and APPI-(MS)_(n), quadrupole mass spectrometry, fouriertransform mass spectrometry (FTMS), and ion trap mass spectrometry,where n is an integer greater than zero.

Detection methods may include use of a biochip array. Biochip arraysuseful in the invention include protein and polynucleotide arrays. Oneor more markers are captured on the biochip array and subjected toanalysis to detect the level of the markers in a sample.

Markers may be captured with capture reagents immobilized to a solidsupport, such as a biochip, a multiwell microtiter plate, a resin, or anitrocellulose membrane that is subsequently probed for the presence orlevel of a marker. Capture can be on a chromatographic surface or abiospecific surface. For example, a sample containing the markers, suchas serum, may be used to contact the active surface of a biochip for asufficient time to allow binding. Unbound molecules are washed from thesurface using a suitable eluant, such as phosphate buffered saline. Ingeneral, the more stringent the eluant, the more tightly the proteinsmust be bound to be retained after the wash.

Upon capture on a biochip, analytes can be detected by a variety ofdetection methods selected from, for example, a gas phase ionspectrometry method, an optical method, an electrochemical method,atomic force microscopy and a radio frequency method. In one embodiment,mass spectrometry, and in particular, SELDI, is used. Optical methodsinclude, for example, detection of fluorescence, luminescence,chemiluminescence, absorbance, reflectance, transmittance, birefringenceor refractive index (e.g., surface plasmon resonance, ellipsometry, aresonant mirror method, a grating coupler waveguide method orinterferometry). Optical methods include microscopy (both confocal andnon-confocal), imaging methods and non-imaging methods. Immunoassays invarious formats (e.g., ELISA) are popular methods for detection ofanalytes captured on a solid phase. Electrochemical methods includevoltametry and amperometry methods. Radio frequency methods includemultipolar resonance spectroscopy.

Mass spectrometry (MS) is a well-known tool for analyzing chemicalcompounds. Thus, in one embodiment, the methods of the present inventioncomprise performing quantitative MS to measure the serum peptide marker.The method may be performed in an automated (Villanueva, et al., NatureProtocols (2006) 1(2):880-891) or semi-automated format. This can beaccomplished, for example with MS operably linked to a liquidchromatography device (LC-MS/MS or LC-MS) or gas chromatography device(GC-MS or GC-MS/MS). Methods for performing MS are known in the fieldand have been disclosed, for example, in US Patent ApplicationPublication Nos: 20050023454; 20050035286; U.S. Pat. No. 5,800,979 andreferences disclosed therein.

The protein fragments, whether they are peptides derived from the mainchain of the protein or are residues of a side-chain, are collected onthe collection layer. They may then be analyzed by a spectroscopicmethod based on matrix-assisted laser desorption/ionization (MALDI) orelectrospray ionization (ESI). The preferred procedure is MALDI withtime of flight (TOF) analysis, known as MALDI-TOF MS. This involvesforming a matrix on the membrane, e.g. as described in the literature,with an agent which absorbs the incident light strongly at theparticular wavelength employed. The sample is excited by UV, or IR laserlight into the vapour phase in the MALDI mass spectrometer. Ions aregenerated by the vaporization and form an ion plume. The ions areaccelerated in an electric field and separated according to their timeof travel along a given distance, giving a mass/charge (m/z) readingwhich is very accurate and sensitive. MALDI spectrometers arecommercially available from PerSeptive Biosystems, Inc. (Frazingham,Mass., USA) and are described in the literature, e.g. M. Kussmann and P.Roepstorff, cited above.

Magnetic-based serum processing can be combined with traditionalMALDI-TOF. Through this approach, improved peptide capture is achievedprior to matrix mixture and deposition of the sample on MALDI targetplates. Accordingly, methods of peptide capture are enhanced through theuse of derivatized magnetic bead based sample processing.

MALDI-TOF MS allows scanning of the fragments of many proteins at once.Thus, many proteins can be run simultaneously on a polyacrylamide gel,subjected to a method of the invention to produce an array of spots onthe collecting membrane, and the array may be analyzed. Subsequently,automated output of the results is provided by using the ExPASy server,as at present used for MIDI-TOF MS and to generate the data in a formsuitable for computers.

Other techniques for improving the mass accuracy and sensitivity of theMALDI-TOF MS can be used to analyze the fragments of protein obtained onthe collection membrane. These include the use of delayed ionextraction, energy reflectors and ion-trap modules. In addition, postsource decay and MS--MS analysis are useful to provide furtherstructural analysis. With ESI, the sample is in the liquid phase and theanalysis can be by ion-trap, TOF, single quadrupole or multi-quadrupolemass spectrometers. The use of such devices (other than a singlequadrupole) allows MS--MS or MS^(n) analysis to be performed. Tandemmass spectrometry allows multiple reactions to be monitored at the sametime.

Capillary infusion may be employed to introduce the marker to a desiredMS implementation, for instance, because it can efficiently introducesmall quantities of a sample into a mass spectrometer without destroyingthe vacuum. Capillary columns are routinely used to interface theionization source of a MS with other separation techniques including gaschromatography (GC) and liquid chromatography (LC). GC and LC can serveto separate a solution into its different components prior to massanalysis. Such techniques are readily combined with MS, for instance.One variation of the technique is that high performance liquidchromatography (HPLC) can now be directly coupled to mass spectrometerfor integrated sample separation/and mass spectrometer analysis.

Quadrupole mass analyzers may also be employed as needed to practice theinvention. Fourier-transform ion cyclotron resonance (FTMS) can also beused for some invention embodiments. It offers high resolution and theability of tandem MS experiments. FTMS is based on the principle of acharged particle orbiting in the presence of a magnetic field. Coupledto ESI and MALDI, FTMS offers high accuracy with errors as low as0.001%.

In one embodiment, the marker qualification methods of the invention mayfurther comprise identifying significant peaks from combined spectra.The methods may also further comprise searching for outlier spectra. Inanother embodiment, the method of the invention further comprisesdetermining distant dependent K-nearest neighbors.

In another embodiment of the method of the invention, an ion mobilityspectrometer can be used to detect and characterize serum peptidemarkers. The principle of ion mobility spectrometry is based ondifferent mobility of ions. Specifically, ions of a sample produced byionization move at different rates, due to their difference in, e.g.,mass, charge, or shape, through a tube under the influence of anelectric field. The ions (typically in the form of a current) areregistered at the detector which can then be used to identify a markeror other substances in a sample. One advantage of ion mobilityspectrometry is that it can operate at atmospheric pressure.

In an additional embodiment of the methods of the present invention,multiple markers are measured. The use of multiple markers increases thepredictive value of the test and provides greater utility in diagnosis,toxicology, patient stratification and patient monitoring. The processcalled “Pattern recognition” detects the patterns formed by multiplemarkers greatly improves the sensitivity and specificity of clinicalproteomics for predictive medicine. Subtle variations in data fromclinical samples indicate that certain patterns of protein expressioncan predict phenotypes such as the presence or absence of a certaindisease, a particular stage of cancer-progression, or a positive oradverse response to drug treatments.

Expression levels of particular nucleic acids or polypeptides arecorrelated with a neoplasia, such as prostate carcinoma, and thus areuseful in diagnosis. Antibodies that bind a polypeptide describedherein, oligonucleotides or longer fragments derived from a nucleic acidsequence described herein (e.g., an OCT3/4, SOX2, Nanog, c-Myc, Klf4,keratin 8, and uPAR nucleic acid sequence), or any other method known inthe art may be used to monitor expression of a polynucleotide orpolypeptide of interest. Detection of an alteration relative to anormal, reference sample can be used as a diagnostic indicator ofprostate carcinoma. In particular embodiments, the expression of aOCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, and uPAR polypeptide isindicative of prostate carcinoma or the propensity to develop prostatecarcinoma. In particular embodiments, the expression of OCT3/4, SOX2,and Nanog polypeptides is indicative of prostate carcinoma or thepropensity to develop prostate carcinoma. In particular embodiments, theexpression of OCT3/4, SOX2, Nanog, and c-Myc polypeptides is indicativeof prostate carcinoma or the propensity to develop prostate carcinoma.In other embodiments, a 2, 3, 4, 5, or 6-fold change in the level of amarker of the invention is indicative of prostate carcinoma. In yetanother embodiment, an expression profile that characterizes alterationsin the expression two or more markers is correlated with a particulardisease state (e.g., prostate carcinoma). Such correlations areindicative of prostate carcinoma or the propensity to develop prostatecarcinoma. Prostate cancers that express increased levels of one or allof OCT3/4, SOX2, Nanog, and c-Myc are identified as in need of immediateor aggressive therapy, whereas prostate cancers that express low orvirtually undetectable levels of one or all of OCT3/4, SOX2, Nanog, andc-Myc are identified as unlikely to metastasize. This molecular profileindicates that the neoplasia may be monitored for weeks, months, oryears prior to therapy. In one embodiment, a prostate carcinoma can bemonitored using the methods and compositions of the invention.

In one embodiment, the level of one or more markers is measured on atleast two different occasions and an alteration in the levels ascompared to normal reference levels over time is used as an indicator ofprostate carcinoma or the propensity to develop prostate carcinoma. Thelevel of marker in the bodily fluids (e.g., blood, blood serum, plasma,saliva, urine, seminal fluids, and ejaculate) of a subject havingprostate carcinoma or the propensity to develop such a condition may bealtered by as little as 10%, 20%, 30%, or 40%, or by as much as 50%,60%, 70%, 80%, or 90% or more relative to the level of such marker in anormal control. In general, levels of OCT3/4, SOX2, Nanog, c-Myc, Klf4,keratin 8, and uPAR are present at low or undetectable levels in ahealthy subject (i.e., those who do not have and/or who will not developprostate carcinoma). In one embodiment, a subject sample of a bodilyfluid (e.g., blood, blood serum, plasma, saliva, urine, seminal fluids,and ejaculate) is collected prior to the onset of symptoms of prostatecarcinoma. In another example, the sample can be a tissue or cellcollected prior to the onset of prostate carcinoma symptoms.

The diagnostic methods described herein can be used individually or incombination with any other diagnostic method described herein for a moreaccurate diagnosis of the presence or severity of prostate carcinoma.

The diagnostic methods described herein can also be used to monitor andmanage prostate carcinoma, or to reliably distinguish prostate carcinomafrom benign prostatic hyperplasia.

As indicated above, the invention provides methods for aiding a humancancer diagnosis using one or more markers, as specified herein. Thesemarkers can be used alone, in combination with other markers in any set,or with entirely different markers in aiding human cancer diagnosis. Themarkers are differentially present in samples of a human cancer patientand a normal subject in whom human cancer is undetectable. Therefore,detection of one or more of these markers in a person would provideuseful information regarding the probability that the person may haveprostate cancer or regarding the aggressiveness of the cancer.

The detection of the peptide marker is then correlated with a probablediagnosis of cancer. In some embodiments, the detection of the merepresence of a marker (e.g., SOX2 OCT3/4, Nanog, and/or c-Myc), withoutquantifying the amount thereof, is useful and can be correlated with aprobable diagnosis of cancer. The measurement of markers may alsoinvolve quantifying the markers to correlate the detection of markerswith a probable diagnosis of cancer. Thus, if the amount of the markersdetected in a subject being tested is different compared to a controlamount (i.e., higher than the control), then the subject being testedhas a higher probability of having cancer.

The correlation may take into account the amount of the marker ormarkers in the sample compared to a control amount of the marker ormarkers (e.g., in normal subjects or in non-cancer subjects such aswhere cancer is undetectable). A control can be, e.g., the average ormedian amount of marker present in comparable samples of normal subjectsin normal subjects or in non-cancer subjects such as where cancer isundetectable. The control amount is measured under the same orsubstantially similar experimental conditions as in measuring the testamount. As a result, the control can be employed as a referencestandard, where the normal (non-cancer) phenotype is known, and eachresult can be compared to that standard, rather than re-running acontrol.

Accordingly, a marker profile may be obtained from a subject sample andcompared to a reference marker profile obtained from a referencepopulation, so that it is possible to classify the subject as belongingto or not belonging to the reference population. The correlation maytake into account the presence or absence of the markers in a testsample and the frequency of detection of the same markers in a control.The correlation may take into account both of such factors to facilitatedetermination of cancer status.

In certain embodiments of the methods of qualifying cancer status, themethods further comprise managing subject treatment based on the status.The invention also provides for such methods where the markers (orspecific combination of markers) are measured again after subjectmanagement. In these cases, the methods are used to monitor the statusof the cancer, e.g., response to cancer treatment, remission of thedisease or progression of the disease.

The markers of the present invention have a number of other uses. Forexample, they can be used to monitor responses to certain treatments ofhuman cancer. In yet another example, the markers can be used inheredity studies. For instance, certain markers may be geneticallylinked. This can be determined by, e.g., analyzing samples from apopulation of human cancer subjects whose families have a history ofcancer. The results can then be compared with data obtained from, e.g.,cancer subjects whose families do not have a history of cancer. Themarkers that are genetically linked may be used as a tool to determineif a subject whose family has a history of cancer is pre-disposed tohaving cancer.

Any marker, individually, is useful in aiding in the determination ofcancer status. First, the selected marker is detected in a subjectsample using the methods described herein. Then, the result is comparedwith a control that distinguishes cancer status from non-cancer status.As is well understood in the art, the techniques can be adjusted toincrease sensitivity or specificity of the diagnostic assay depending onthe preference of the diagnostician.

While individual markers are useful diagnostic markers, in someinstances, a combination of markers provides greater predictive valuethan single markers alone. The detection of a plurality of markers (orabsence thereof, as the case may be) in a sample can increase thepercentage of true positive and true negative diagnoses and decrease thepercentage of false positive or false negative diagnoses. Thus,preferred methods of the present invention comprise the measurement ofmore than one marker.

Microarrays

As reported herein, a number of markers (e.g., OCT3/4, SOX2, Nanog,c-Myc, Klf4, keratin 8, and uPAR) have been identified that areassociated with neoplasia, such as prostate carcinoma. Methods forassaying the expression of these polypeptides are useful forcharacterizing the neoplasia (e.g., prostate carcinoma). In particular,the invention provides diagnostic methods and compositions useful foridentifying a polypeptide expression profile that identifies a subjectas having or having a propensity to develop a neoplasia (e.g., prostatecarcinoma). Such assays can be used to measure an alteration in thelevel of a polypeptide.

The polypeptides and nucleic acid molecules of the invention are usefulas hybridizable array elements in a microarray. The array elements areorganized in an ordered fashion such that each element is present at aspecified location on the substrate. Useful substrate materials includemembranes, composed of paper, nylon or other materials, filters, chips,glass slides, and other solid supports. The ordered arrangement of thearray elements allows hybridization patterns and intensities to beinterpreted as expression levels of particular genes or proteins.Methods for making nucleic acid microarrays are known to the skilledartisan and are described, for example, in U.S. Pat. No. 5,837,832,Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al.(Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated byreference. Methods for making polypeptide microarrays are described, forexample, by Ge (Nucleic Acids Res. 28: e3. i-e3. vii, 2000), MacBeath etal., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet.26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated byreference.

Protein Microarrays

Proteins (e.g., OCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, and uPAR)may be analyzed using protein microarrays. Such arrays are useful inhigh-throughput low-cost screens to identify alterations in theexpression or post-translation modification of a polypeptide of theinvention, or a fragment thereof. In particular, such microarrays areuseful to identify a protein whose expression is altered in prostatecarcinoma. In one embodiment, a protein microarray of the inventionbinds a marker present in a subject sample and detects an alteration inthe level of the marker. Typically, a protein microarray features aprotein, or fragment thereof, bound to a solid support. Suitable solidsupports include membranes (e.g., membranes composed of nitrocellulose,paper, or other material), polymer-based films (e.g., polystyrene),beads, or glass slides. For some applications, proteins (e.g.,antibodies that bind a marker of the invention) are spotted on asubstrate using any convenient method known to the skilled artisan(e.g., by hand or by inkjet printer).

The protein microarray is hybridized with a detectable probe. Suchprobes can be polypeptide, nucleic acid molecules, antibodies, or smallmolecules. For some applications, polypeptide and nucleic acid moleculeprobes are derived from a biological sample taken from a patient; suchas a bodily fluid (such as blood, blood serum, plasma, saliva, urine,seminal fluids, and ejaculate); a homogenized tissue sample (e.g. atissue sample obtained by biopsy); or a cell isolated from a patientsample. Probes can also include antibodies, candidate peptides, nucleicacids, or small molecule compounds derived from a peptide, nucleic acid,or chemical library. Hybridization conditions (e.g., temperature, pH,protein concentration, and ionic strength) are optimized to promotespecific interactions. Such conditions are known to the skilled artisanand are described, for example, in Harlow, E. and Lane, D., UsingAntibodies: A Laboratory Manual. 1998, New York: Cold Spring HarborLaboratories. After removal of non-specific probes, specifically boundprobes are detected, for example, by fluorescence, enzyme activity(e.g., an enzyme-linked calorimetric assay), direct immunoassay,radiometric assay, or any other suitable detectable method known to theskilled artisan.

Nucleic Acid Microarrays

To produce a nucleic acid microarray, oligonucleotides may besynthesized or bound to the surface of a substrate using a chemicalcoupling procedure and an ink jet application apparatus, as described inPCT application WO95/251116 (Baldeschweiler et al.), incorporated hereinby reference. Alternatively, a gridded array may be used to arrange andlink cDNA fragments or oligonucleotides to the surface of a substrateusing a vacuum system, thermal, UV, mechanical or chemical bondingprocedure.

A nucleic acid molecule (e.g. RNA or DNA) derived from a biologicalsample may be used to produce a hybridization probe as described herein.The biological samples are generally derived from a patient, preferablyas a bodily fluid (such as blood, blood serum, plasma, saliva, urine,seminal fluids, and ejaculate) or tissue sample (e.g. a tissue sampleobtained by biopsy). For some applications, cultured cells or othertissue preparations may be used. The mRNA is isolated according tostandard methods, and cDNA is produced and used as a template to makecomplementary RNA suitable for hybridization. Such methods are known inthe art. The RNA is amplified in the presence of fluorescentnucleotides, and the labeled probes are then incubated with themicroarray to allow the probe sequence to hybridize to complementaryoligonucleotides bound to the microarray.

Incubation conditions are adjusted such that hybridization occurs withprecise complementary matches or with various degrees of lesscomplementarity depending on the degree of stringency employed. Forexample, stringent salt concentration will ordinarily be less than about750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500mM NaCl and 50 mM trisodium citrate, and most preferably less than about250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridizationcan be obtained in the absence of organic solvent, e.g., formamide,while high stringency hybridization can be obtained in the presence ofat least about 35% formamide, and most preferably at least about 50%formamide. Stringent temperature conditions will ordinarily includetemperatures of at least about 30 C., more preferably of at least about37 C., and most preferably of at least about 42 C. Varying additionalparameters, such as hybridization time, the concentration of detergent,e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion ofcarrier DNA, are well known to those skilled in the art. Various levelsof stringency are accomplished by combining these various conditions asneeded. In a preferred embodiment, hybridization will occur at 30 C in750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferredembodiment, hybridization will occur at 37 C. in 500 mM NaCl, 50 mMtrisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmonsperm DNA (ssDNA). In a most preferred embodiment, hybridization willoccur at 42 C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50%formamide, and 200 μg/ml ssDNA. Useful variations on these conditionswill be readily apparent to those skilled in the art.

The removal of nonhybridized probes may be accomplished, for example, bywashing. The washing steps that follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude a temperature of at least about 25 C., more preferably of atleast about 42.degree. C., and most preferably of at least about 68 C.In a preferred embodiment, wash steps will occur at 25 C in 30 mM NaCl,3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment,wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate,and 0.1% SDS. In a most preferred embodiment, wash steps will occur at68 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

A detection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct nucleic acid sequencessimultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci.94:2150-2155, 1997). Preferably, a scanner is used to determine thelevels and patterns of fluorescence.

Diagnostic Kits

The invention provides kits for diagnosing or monitoring a neoplasia,such as a prostate carcinoma, or for selecting a treatment for aneoplasia (e.g., prostate carcinoma). In one embodiment, the kitincludes a composition containing at least one agent that binds apolypeptide or polynucleotide (e.g., any one or more of OCT3/4, Nanog;Sox2, c-Myc, Klf4, Keratin 8, and uPAR) whose expression is increased inprostate carcinoma. In one embodiment, the kit contains agents that bindOCT3/4, SOX2, Nanog, and/or c-Myc. In another embodiment, the inventionprovides a kit that contains an agent that binds a nucleic acid moleculewhose expression is altered in a neoplasia (e.g., prostate carcinoma).If desired, a kit of the invention comprises an agent (e.g., anantibody, aptamer, or other agent) that binds E-cadherin. Such agentsmay be used to select cells that bind E-cadherin from a biologicalsample derived from a subject. Cells selected as binding E-cadherin arethen analyzed to determine the level of any one or more of a marker ofthe invention expressed by the cells (e.g., OCT3/4, Nanog, Sox2, c-Myc,Klf4, Keratin 8, and/or uPAR). In some embodiments, the kit comprises asterile container which contains the binding agent; such containers canbe boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs,or other suitable container forms known in the art. Such containers canbe made of plastic, glass, laminated paper, metal foil, or othermaterials suitable for holding medicaments.

If desired the kit is provided together with instructions for using thekit to diagnose a neoplasia (e.g., prostate carcinoma). The instructionswill generally include information about the use of the composition fordiagnosing a subject as having a neoplasia (e.g., prostate carcinoma) orhaving a propensity to develop a neoplasia (e.g., prostate carcinoma).In other embodiments, the instructions include at least one of thefollowing: description of the binding agent; warnings; indications;counter-indications; animal study data; clinical study data; and/orreferences. The instructions may be printed directly on the container(when present), or as a label applied to the container, or as a separatesheet, pamphlet, card, or folder supplied in or with the container.

Subject Monitoring

The disease state or treatment of a subject having a neoplasia, benignprostatic hyperplasia, prostate carcinoma, or a propensity to developsuch a condition can be monitored using the methods and compositions ofthe invention. In other embodiments, compositions and methods of theinvention are used by a clinician to identify subjects as having or nothaving a neoplasia (e.g., prostate cancer). For example, a generalpractitioner may use the methods delineated herein to screen patientsfor the presence of a neoplasia or for prostate cancer. In oneembodiment, the expression of markers present in a bodily fluid, such asblood, blood serum, plasma, saliva, urine, seminal fluids, andejaculate, is monitored. Such monitoring may be useful, for example, inassessing the efficacy of a particular drug in a subject or in assessingdisease progression. Therapeutics that decrease the expression of amarker of the invention (e.g., OCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin8, and/or uPAR) are taken as particularly useful in the invention.

Types of Biological Samples

The level of OCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, and/or uPARprotein or polynucleotide is measured in different types of biologicsamples. In one embodiment, the level of OCT3/4, SOX2, and Nanogproteins or polynucleotides is measured in a biologic sample. In anotherembodiment, the level of OCT3/4, SOX2, Nanog, and c-Myc proteins orpolynucleotides is measured in a biologic sample. In one embodiment, thebiologic sample is a tissue sample that includes cells of a tissue ororgan (e.g., prostatic tissue cells). Prostatic tissue is obtained, forexample, from a biopsy of the prostate. In another embodiment, thebiologic sample is a biologic fluid sample. Biological fluid samplesinclude blood, blood serum, plasma, saliva, urine, seminal fluids, andejaculate, or any other biological fluid useful in the methods of theinvention.

Diagnostic Assays

The present invention provides a number of diagnostic assays that areuseful for the identification or characterization of a neoplasia, abenign prostatic hyperplasia, prostate carcinoma, or a propensity todevelop such a condition. In one embodiment, prostate carcinoma ischaracterized by quantifying the level of one or more of the followingmarkers: OCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, and uPAR. Inanother embodiment, prostate carcinoma is characterized by quantifyingthe level of one or more of the following markers: OCT3/4, SOX2, andNanog. In yet another embodiment, prostate carcinoma is characterized byquantifying the level of the following markers: OCT3/4, SOX2, Nanog, andc-Myc. While the examples provided below describe specific methods ofdetecting levels of these markers, the skilled artisan appreciates thatthe invention is not limited to such methods. Marker levels arequantifiable by any standard method, such methods include, but are notlimited to real-time PCR, Southern blot, PCR, mass spectroscopy, and/orantibody binding.

The examples describe primers used in the invention for amplification ofmarkers of the invention. The primers of the invention embraceoligonucleotides of sufficient length and appropriate sequence so as toprovide specific amplification. While exemplary primers are providedherein, it is understood that any primer that hybridizes with the markersequences of the invention are useful in the methods of the inventionfor detecting marker levels.

The level of any two or more of the markers described herein defines themarker profile of a prostate carcinoma. The level of marker is comparedto a reference. In one embodiment, the reference is the level of markerpresent in a control sample obtained from a patient that does not haveprostate carcinoma. In another embodiment, the reference is a baselinelevel of marker present in a biologic sample derived from a patientprior to, during, or after treatment for a neoplasia. In yet anotherembodiment, the reference is a standardized curve. The level of any oneor more of the markers described herein (e.g., the combination ofOCT3/4, SOX2, Nanog, c-Myc, Klf4, keratin 8, and uPAR; the combinationof OCT3/4, SOX2, and Nanog; the combination of OCT3/4, SOX2, Nanog, andc-Myc) is used, alone or in combination with other standard methods, todetermine the stage or grade of a neoplasia. Grading is used to describehow abnormal or aggressive the neoplastic cells appear, while staging isused to describe the extent of the neoplasia. The grade and stage of theneoplasia is indicative of the patient's long-term prognosis (i.e.,probable response to treatment and survival). Thus, the methods of theinvention are useful for predicting a patient's prognosis, and forselecting a course of treatment.

The Gleason scale is the most common scale used for grading prostatecancer. A pathologist will look at the two most poorly differentiatedparts of the tumor and grade them. The Gleason score is the sum of thetwo grades, and so can range from two to 10. The higher the score is,the poorer the prognosis. Scores usually range between 4 and 7. Thescores can be broken down into three general categories: (i) low-gradeneoplasias (score≦4) are typically slow-growing and contain cells thatare most similar to normal prostate cells; intermediate grade neoplasias(4<score≦7) are the most common and typically contain some cells thatare similar to normal prostate cells as well as some more abnormalcells; high-grade neoplasias (8≦score≦10) contain cells that are mostdissimilar to normal prostate cells. High-grade neoplasias are the mostdeadly because they are most aggressive and fast growing. High-gradeneoplasias typically move rapidly into surrounding tissues, such aslymph nodes and bones.

Stage refers to the extent of a cancer. In prostate cancer, for example,one staging method divides the cancer into four categories, A, B, C, andD. Stage A describes a cancer that is only found by elevated PSA andbiopsy, or at surgery for obstruction. It is not palpable on digitalrectal exam (DRE). This stage is localized to the prostate. This type ofcancer is usually curable, especially if it has a relatively low Gleasongrade. Stage B refers to a cancer that can be felt on rectal examinationand is limited to the prostate. Bone scans or CT/MRI scans are oftenused to determine this stage, particularly if prostate specific antigen(PSA) levels are significantly elevated or if the Gleason grade is 7 orgreater. Many Stage B prostate cancers are curable. Stage C cancers havespread beyond the capsule of the prostate into local organs or tissues,but have not yet metastasized to other sites. This stage is determinedby DRE, or CT/MRI scans, and/or sonography. In Stage C a bone scan or aPROSTASCINT scan is negative. Some Stage C cancers are curable. Stage Dcancer has metastasized to distant lymph nodes, bones or other sites.This is usually determined by bone scan, PROSTASCINT scan, or otherstudies. Stage D cancer is usually incurable, but may be treatable.

Selection of a Treatment Method

After a subject is diagnosed as having a neoplasia (e.g., prostatecarcinoma) a method of treatment is selected. In prostate cancer, forexample, a number of standard treatment regimens are available. Themarker profile of the neoplasia is used in selecting a treatment method.In one embodiment, less aggressive neoplasias have lower levels of SOX2than more aggressive neoplasias. In another embodiment, the markerprofile of a neoplasia, or the level of SOX2 in the neoplasia iscorrelated with a clinical outcome using statistical methods todetermine the aggressiveness of the neoplasia. Prostate carcinomashaving increased levels of SOX2, alone or in combination with increasedlevels of OCT3/4, Nanog, c-Myc, Klf4, keratin 8, and uPAR, have a markerprofile that correlates with a poor clinical outcome, such as metastasisor death. Prostate carcinomas having increased levels of SOX2, alone orin combination with increased levels of OCT3/4 and Nanog, or OCT3/4,Nanog, and c-Myc, have a marker profile that correlates with a poorclinical outcome, such as metastasis or death. Such prostate carcinomasare identified as aggressive neoplasias. Marker profiles (e.g., prostatecarcinomas that fail to express detectable levels of SOX2, and/orprostate carcinomas that fail to express detectable levels of one ormore of OCT3/4, Nanog, c-Myc, Klf4, keratin 8, and uPAR) that correlatewith good clinical outcomes are identified as less aggressiveneoplasias.

Less aggressive neoplasias are likely to be susceptible to conservativetreatment methods. Conservative treatment methods include, for example,cancer surveillance, which involves periodic patient monitoring usingdiagnostic assays of the invention, alone or in combination, with PSAblood tests and DREs, or hormonal therapy. Cancer surveillance isselected when diagnostic assays indicate that the adverse effects oftreatment (e.g., impotence, urinary, and bowel disorders) are likely tooutweigh therapeutic benefits.

More aggressive neoplasias are identified as having increased levels ofSox2 relative to corresponding control cells. Such neoplasias are lesssusceptible to conservative treatment methods. When methods of theinvention indicate that a neoplasia is very aggressive, an aggressivemethod of treatment should be selected. Aggressive therapeutic regimenstypically include one or more of the following therapies: radicalprostatectomy, radiation therapy (e.g., external beam andbrachytherapy), hormone therapy, and chemotherapy.

Patient Monitoring

The diagnostic methods of the invention are also useful for monitoringthe course of a neoplasia (e.g., prostate carcinoma) in a patient or forassessing the efficacy of a therapeutic regimen. In one embodiment, thediagnostic methods of the invention are used periodically to monitor thepolynucleotide or polypeptide levels of one or more of OCT3/4, SOX2,Nanog, c-Myc, Klf4, keratin 8, and uPAR. In another embodiment, thediagnostic methods of the invention are used periodically to monitor thepolynucleotide or polypeptide levels of OCT3/4, SOX2, and Nanog. In yetanother embodiment, the diagnostic methods of the invention are usedperiodically to monitor the polynucleotide or polypeptide levels ofOCT3/4, SOX2, Nanog, and c-Myc. In one example, the neoplasia ischaracterized using a diagnostic assay of the invention prior toadministering therapy. This assay provides a baseline that describes thelevel of one or more markers of the neoplasia prior to treatment.Additional diagnostic assays are administered during the course oftherapy to monitor the efficacy of a selected therapeutic regimen. Atherapy is identified as efficacious when a diagnostic assay of theinvention detects a decrease in marker levels relative to the baselinelevel of marker prior to treatment.

Cancer Stem Cells

The development of human neoplasia (e.g., prostate cancer) proceedsthrough a series of defined stages, beginning with prostaticintraepithelial neoplasia, progressing to invasive hormone-dependentcancer, and finally progressing to hormone-independent cancer. Mosthuman prostate cancers are adenocarcinomas that express markersassociated with luminal epithelial cells. Because of unbalanced cellproliferation, cell differentiation, and cell death, prostate cancerexhibits substantial histological heterogeneity. To date, DNA and tissuemicroarrays of tumors have failed to account for cellular heterogeneityand differences in the proliferative potential of different populationswithin tumors. At present, all of the phenotypically diverse cancercells are treated as though they have unlimited proliferative potentialand can acquire the ability to metastasize. In patients with metasticdisease, conventional therapies are ineffective. Metastatic prostatetumor cells are able to survive extreme conditions within thecirculation. Metastic cancer cells lodge in the capillary beds ofdistant organs where they undergo extensive proliferation, often inbone, lymph node, lung and brain. Metastatic tumor cells share manycharacteristics (e.g., self-renewal, proliferation, and multi-potency)with pluripotent stem cells. Little is known about how human metastatictumor cells maintain or acquire their multipotency. Recent studiessuggest the existence of prostate cancer stem cells that arechemo-resistant and radiation-resistant. Therapies specifically directedagainst such cancer stem cells are likely to be more effective in curingprostate cancer and metastatic disease.

Accordingly, the present invention provides methods of treatingneoplasia (e.g., prostate cancer) and/or disorders or symptoms thereofwhich comprise administering a therapeutically effective amount of apharmaceutical composition comprising an agent of the formulae herein toa subject (e.g., a mammal, such as a human). Thus, one embodiment is amethod of treating a subject suffering from or susceptible to prostatecancer, metastatic prostate cancer, or prostate cancer having thepropensity to metastasize or symptoms thereof. The method includes thestep of administering to the mammal a therapeutic amount of an agentherein sufficient to treat the prostate cancer or symptom thereof, underconditions such that the prostate cancer is treated.

The methods herein include administering to the subject (including asubject identified as in need of such treatment) an effective amount ofa compound described herein, or a composition described herein toproduce such effect. Identifying a subject in need of such treatment canbe in the judgment of a subject or a health care professional and can besubjective (e.g. opinion) or objective (e.g. measurable by a test ordiagnostic method).

The therapeutic methods of the invention (which include prophylactictreatment) in general comprise administration of a therapeuticallyeffective amount of the agents herein, such as an agent of the formulaeherein to a subject (e.g., animal, human) in need thereof, including amammal, particularly a human. Such treatment will be suitablyadministered to subjects, particularly humans, suffering from, having,susceptible to, or at risk for prostate cancer, including metastaticdisease or prostate cancer having a propensity to metastasize, or asymptom thereof. Determination of those subjects “at risk” can be madeby any objective or subjective determination by a diagnostic test oropinion of a subject or health care provider (e.g., genetic test, enzymeor protein marker, Marker (as defined herein), family history, and thelike). The compounds herein may be also used in the treatment of anyother disorders in which prostate cancer or hyperplasia may beimplicated.

In one embodiment, the invention provides a method of monitoringtreatment progress. The method includes the step of determining a levelof diagnostic marker (Marker) (e.g., Sox2, alone or in combination withOCT3/4, Nanog, c-Myc, Klf4, Keratin 8, and uPAR or any target delineatedherein modulated by a compound herein, a protein or indicator thereof,etc.) or diagnostic measurement (e.g., screen, assay) in a subjectsuffering from or susceptible to prostate cancer, in which the subjecthas been administered a therapeutic amount of a compound hereinsufficient to treat the disease or symptoms thereof. The level of Markerdetermined in the method can be compared to known levels of Marker ineither healthy normal controls or in other afflicted patients toestablish the subject's disease status. In preferred embodiments, asecond level of Marker in the subject is determined at a time pointlater than the determination of the first level, and the two levels arecompared to monitor the course of disease or the efficacy of thetherapy. In certain preferred embodiments, a pre-treatment level ofMarker in the subject is determined prior to beginning treatmentaccording to this invention; this pre-treatment level of Marker can thenbe compared to the level of Marker in the subject after the treatmentcommences, to determine the efficacy of the treatment.

Therapeutic Uses

The present invention features methods for treating neoplasia (e.g.,prostate cancer) or the progression of a neoplasia, such as prostate,cancer by administering OCT3/4, NANOG, SOX2, C-MYC or KLF4 inhibitorynucleic acid molecules or agents that decrease the expression orbiological activity of an OCT3/4, NANOG, SOX2, C-MYC or KLF4 nucleicacid molecule or polypeptide. In other embodiments, the method involvesadministering an inhibitory nucleic acid molecule or other agent thatdecreases the expression or biological activity of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and uPAR, that decreases the expression orbiological activity of each of Sox2, Oct3/4, and Nanog, or thatdecreases the expression or biological activity of Sox2 alone, or incombination with any other marker described herein. Advantageously, suchagents selectively target prostate tumor initiating stem cells.Compounds of the present invention may be administered by anyappropriate route for the treatment or prevention of neoplasia. Thesemay be administered to humans, domestic pets, livestock, or otheranimals with a pharmaceutically acceptable diluent, carrier, orexcipient, in unit dosage form. Administration may be parenteral,intravenous, intra-arterial, subcutaneous, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal,intracisternal, intraperitoneal, intranasal, aerosol, by suppositories,or oral administration.

Therapeutic formulations may be in the form of liquid solutions orsuspensions; for oral administration, formulations may be in the form oftablets or capsules; and for intranasal formulations, in the form ofpowders, nasal drops, or aerosols.

Methods well known in the art for making formulations are found, forexample, in Remington: The Science and Practice of Pharmacy (20th ed.,ed. A. R. Gennaro, 2000, Lippincott Williams & Wilkins). Formulationsfor parenteral administration may, for example, contain excipients,sterile water, or saline, polyalkylene glycols such as polyethyleneglycol, oils of vegetable origin, or hydrogenated napthalenes.Biocompatible, biodegradable lactide polymer, lactide/glycolidecopolymer, or polyoxyethylene-polyoxypropylene copolymers may be used tocontrol the release of the compounds. Nanoparticulate formulations(e.g., biodegradable nanoparticles, solid lipid nanoparticles,liposomes) may be used to control the biodistribution of the compounds.Other potentially useful parenteral delivery systems includeethylene-vinyl acetate copolymer particles, osmotic pumps, implantableinfusion systems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycholate anddeoxycholate, or may be oily solutions for administration in the form ofnasal drops, or as a gel. The concentration of the compound in theformulation will vary depending upon a number of factors, including thedosage of the drug to be administered, and the route of administration.

The compound may be optionally administered as a pharmaceuticallyacceptable salt, such as a non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

Administration of compounds in controlled release formulations is usefulwhere the compound of formula I has (i) a narrow therapeutic index(e.g., the difference between the plasma concentration leading toharmful side effects or toxic reactions and the plasma concentrationleading to a therapeutic effect is small; generally, the therapeuticindex, TI, is defined as the ratio of median lethal dose (LD50) tomedian effective dose (ED50)); (ii) a narrow absorption window in thegastro-intestinal tract; or (iii) a short biological half-life, so thatfrequent dosing during a day is required in order to sustain the plasmalevel at a therapeutic level.

Many strategies can be pursued to obtain controlled release in which therate of release outweighs the rate of metabolism of the therapeuticcompound. For example, controlled release can be obtained by theappropriate selection of formulation parameters and ingredients,including, e.g., appropriate controlled release compositions andcoatings. Examples include single or multiple unit tablet or capsulecompositions, oil solutions, suspensions, emulsions, microcapsules,microspheres, nanoparticles, patches, and liposomes.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andantiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

Inhibitory Nucleic Acids

Inhibitory nucleic acid molecules are those oligonucleotides thatinhibit the expression or activity of a OCT3/4, Nanog, Sox2, c-Myc,Klf4, Keratin 8, or uPAR polypeptide. Such oligonucleotides includesingle and double stranded nucleic acid molecules (e.g., DNA, RNA, andanalogs thereof) that bind a nucleic acid molecule that encodes aOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR polypeptide (e.g.,antisense molecules, siRNA, shRNA) as well as nucleic acid moleculesthat bind directly to a OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, oruPAR polypeptide or polynucleotide to modulate its biological activity(e.g., aptamers).

Ribozymes

Catalytic RNA molecules or ribozymes that include an antisense OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR sequence of the presentinvention can be used to inhibit expression of a OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, or uPAR nucleic acid molecule in vivo. Theinclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theconstructs. The design and use of target RNA-specific ribozymes isdescribed in Haseloff et al., Nature 334:585-591. 1988, and U.S. PatentApplication Publication No. 2003/0003469 A1, each of which isincorporated by reference.

Accordingly, the invention also features a catalytic RNA molecule thatincludes, in the binding arm, an antisense RNA having between eight andnineteen consecutive nucleobases. In preferred embodiments of thisinvention, the catalytic nucleic acid molecule is formed in a hammerheador hairpin motif Examples of such hammerhead motifs are described byRossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Exampleof hairpin motifs are described by Hampel et al., “RNA Catalyst forCleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is acontinuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988,Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al.,Nucleic Acids Research, 18: 299, 1990. These specific motifs are notlimiting in the invention and those skilled in the art will recognizethat all that is important in an enzymatic nucleic acid molecule of thisinvention is that it has a specific substrate binding site which iscomplementary to one or more of the target gene RNA regions, and that ithave nucleotide sequences within or surrounding that substrate bindingsite which impart an RNA cleaving activity to the molecule.

Small hairpin RNAs consist of a stem-loop structure with optional 3′UU-overhangs. While there may be variation, stems can range from 21 to31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp(desirably 4 to 23 bp). For expression of shRNAs within cells, plasmidvectors containing either the polymerase III H1-RNA or U6 promoter, acloning site for the stem-looped RNA insert, and a 4-5-thymidinetranscription termination signal can be employed. The Polymerase IIIpromoters generally have well-defined initiation and stop sites andtheir transcripts lack poly(A) tails. The termination signal for thesepromoters is defined by the polythymidine tract, and the transcript istypically cleaved after the second uridine. Cleavage at this positiongenerates a 3′ UU overhang in the expressed shRNA, which is similar tothe 3′ overhangs of synthetic siRNAs. Additional methods for expressingthe shRNA in mammalian cells are described in the references citedabove.

siRNA

Short twenty-one to twenty-five nucleotide double-stranded RNAs areeffective at down-regulating gene expression (Zamore et al., Cell 101:25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporatedby reference). The therapeutic effectiveness of an siRNA approach inmammals was demonstrated in vivo by McCaffrey et al. (Nature 418:38-39.2002).

Given the sequence of a target gene, siRNAs may be designed toinactivate that gene. Such siRNAs, for example, could be administereddirectly to an affected tissue, or administered systemically. Thenucleic acid sequence of an OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8,or uPAR gene can be used to design small interfering RNAs (siRNAs). The21 to 25 nucleotide siRNAs may be used, for example, as therapeutics totreat a vascular disease or disorder.

The inhibitory nucleic acid molecules of the present invention may beemployed as double-stranded RNAs for RNA interference (RNAi)-mediatedknock-down of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPARexpression. In one embodiment, OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin8, or uPAR expression is reduced in an endothelial cell or an astrocyte.RNAi is a method for decreasing the cellular expression of specificproteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001;Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, 10, Curr.Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251,2002). The introduction of siRNAs into cells either by transfection ofdsRNAs or through expression of siRNAs using a plasmid-based expressionsystem is increasingly being used to create loss-of-function phenotypesin mammalian cells.

In one embodiment of the invention, double-stranded RNA (dsRNA) moleculeis made that includes between eight and nineteen consecutive nucleobasesof a nucleobase oligomer of the invention. The dsRNA can be two distinctstrands of RNA that have duplexed, or a single RNA strand that hasself-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or22 base pairs, but may be shorter or longer (up to about 29 nucleobases)if desired. dsRNA can be made using standard techniques (e.g., chemicalsynthesis or in vitro transcription). Kits are available, for example,from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods forexpressing dsRNA in mammalian cells are described in Brummelkamp et al.Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958,2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc.Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad.Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol.20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002,each of which is hereby incorporated by reference.

Small hairpin RNAs consist of a stem-loop structure with optional 3′UU-overhangs. While there may be variation, stems can range from 21 to31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp(desirably 4 to 23 bp). For expression of shRNAs within cells, plasmidvectors containing either the polymerase III H1-RNA or U6 promoter, acloning site for the stem-looped RNA insert, and a 4-5-thymidinetranscription termination signal can be employed. The Polymerase IIIpromoters generally have well-defined initiation and stop sites andtheir transcripts lack poly(A) tails. The termination signal for thesepromoters is defined by the polythymidine tract, and the transcript istypically cleaved after the second uridine. Cleavage at this positiongenerates a 3′ UU overhang in the expressed shRNA, which is similar tothe 3′ overhangs of synthetic siRNAs. Additional methods for expressingthe shRNA in mammalian cells are described in the references citedabove.

Delivery of Nucleobase Oligomers

Naked inhibitory nucleic acid molecules, or analogs thereof, are capableof entering mammalian cells and inhibiting expression of a gene ofinterest. Nonetheless, it may be desirable to utilize a formulation thataids in the delivery of oligonucleotides or other nucleobase oligomersto cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992,6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is herebyincorporated by reference).

Assays for Measuring Cell Viability

Assays for measuring cell viability are known in the art, and aredescribed, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8);Kangas et al. (Med. Biol. 62, 338-43, 1984); Lundin et al., (Meth.Enzymol. 133, 27-42, 1986); Petty et al. (Comparison of J. Biolum.Chemilum. 10, 29-34, .1995); and Cree et al. (AntiCancer Drugs 6:398-404, 1995). Cell viability can be assayed using a variety ofmethods, including MTT(3-(4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop,Bioorg. & Med. Chem. Lett. 1: 611, 1991; Cory et al., Cancer Comm. 3,207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays forcell viability are also available commercially. These assays include butare not limited to CELLTITER-GLO® Luminescent Cell Viability Assay(Promega), which uses luciferase technology to detect ATP and quantifythe health or number of cells in culture, and the CellTiter-Glo®Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH)cytotoxicity assay (Promega).

Candidate compounds that induce or increase neoplastic cell death (e.g.,increase apoptosis, reduce cell survival) are also useful asanti-neoplasm therapeutics. Assays for measuring cell apoptosis areknown to the skilled artisan. Apoptotic cells are characterized bycharacteristic morphological changes, including chromatin condensation,cell shrinkage and membrane blebbing, which can be clearly observedusing light microscopy. The biochemical features of apoptosis includeDNA fragmentation, protein cleavage at specific locations, increasedmitochondrial membrane permeability, and the appearance ofphosphatidylserine on the cell membrane surface. Assays for apoptosisare known in the art. Exemplary assays include TUNEL (Terminaldeoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays,caspase activity (specifically caspase-3) assays, and assays forfas-ligand and annexin V. Commercially available products for detectingapoptosis include, for example, Apo-ONE® Homogeneous Caspase-3/7 Assay,FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, Calif.), theApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, Calif.), andthe Quick Apoptotic DNA Ladder Detection Kit (BIOVISION, Mountain View,Calif.).

Neoplastic cells have a propensity to metastasize, or spread, from theirlocus of origination to distant points throughout the body. Assays formetastatic potential or invasiveness are known to the skilled artisan.Such assays include in vitro assays for loss of contact inhibition (Kimet al., Proc Natl Acad Sci USA. 101:16251-6, 2004), increased soft agarcolony formation in vitro (Zhong et al., Int J. Oncol. 24(6):1573-9,2004), pulmonary metastasis models (Datta et al., In Vivo, 16:451-7,2002) and Matrigel-based cell invasion assays (Hagemann et al.Carcinogenesis. 25: 1543-1549, 2004). In vivo screening methods for cellinvasiveness are also known in the art, and include, for example,tumorigenicity screening in athymic nude mice. A commonly used in vitroassay to evaluate metastasis is the Matrigel-Based Cell Invasion Assay(BD Bioscience, Franklin Lakes, N.J.).

If desired, candidate compounds selected using any of the screeningmethods described herein are tested for their efficacy using animalmodels of neoplasia. In one embodiment, mice are injected withneoplastic human cells. The mice containing the neoplastic cells arethen injected (e.g., intraperitoneally) with vehicle (PBS) or candidatecompound daily for a period of time to be empirically determined. Miceare then euthanized and the neoplastic tissues are collected andanalyzed for OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR mRNAor protein levels using methods described herein. Compounds thatdecrease OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR mRNA orprotein expression relative to control levels are expected to beefficacious for the treatment of a neoplasm in a subject (e.g., a humanpatient). In another embodiment, the effect of a candidate compound ontumor load is analyzed in mice injected with a human neoplastic cell.The neoplastic cell is allowed to grow to form a mass. The mice are thentreated with a candidate compound or vehicle (PBS) daily for a period oftime to be empirically determined. Mice are euthanized and theneoplastic tissue is collected. The mass of the neoplastic tissue inmice treated with the selected candidate compounds is compared to themass of neoplastic tissue present in corresponding control mice.

Therapy

Therapy may be provided wherever cancer therapy is performed: at home,the doctor's office, a clinic, a hospital's outpatient department, or ahospital. Treatment generally begins at a hospital so that the doctorcan observe the therapy's effects closely and make any adjustments thatare needed. The duration of the therapy depends on the kind of cancerbeing treated, the age and condition of the patient, the stage and typeof the patient's disease, and how the patient's body responds to thetreatment. Drug administration may be performed at different intervals(e.g., daily, weekly, or monthly). Therapy may be given in on-and-offcycles that include rest periods so that the patient's body has a chanceto build healthy new cells and regain its strength.

Depending on the type of cancer and its stage of development, thetherapy can be used to slow the spreading of the cancer, to slow thecancer's growth, to kill or arrest cancer cells that may have spread toother parts of the body from the original tumor, to relieve symptomscaused by the cancer, or to prevent cancer in the first place. As usedherein, the term “prostate cancer” is meant a collection of prostatecells multiplying in an abnormal manner. Cancer growth is uncontrolledand progressive, and occurs under conditions that would not elicit, orwould cause cessation of, multiplication of normal cells.

A nucleobase oligomer of the invention, or other negative regulator ofOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR, may beadministered within a pharmaceutically-acceptable diluent, carrier, orexcipient, in unit dosage form. Conventional pharmaceutical practice maybe employed to provide suitable formulations or compositions toadminister the compounds to patients suffering from a disease that iscaused by excessive cell proliferation. Administration may begin beforethe patient is symptomatic. Any appropriate route of administration maybe employed, for example, administration may be parenteral, intravenous,intraarterial, subcutaneous, intratumoral, intramuscular, intracranial,intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular,intrathecal, intracisternal, intraperitoneal, intranasal, aerosol,suppository, or oral administration. For example, therapeuticformulations may be in the form of liquid solutions or suspensions; fororal administration, formulations may be in the form of tablets orcapsules; and for intranasal formulations, in the form of powders, nasaldrops, or aerosols. Methods well known in the art for makingformulations are found, for example, in “Remington: The Science andPractice of Pharmacy” Ed. A. R. Gennaro, Lippincourt Williams & Wilkins,Philadelphia, Pa., 2000. Formulations for parenteral administration may,for example, contain excipients, sterile water, or saline, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin, orhydrogenated napthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds. Otherpotentially useful parenteral delivery systems for delivering an agentthat disrupts the activity of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin8, or uPAR polypeptides or polynucleotides include ethylene-vinylacetate copolymer particles, osmotic pumps, implantable infusionsystems, and liposomes. Formulations for inhalation may containexcipients, for example, lactose, or may be aqueous solutionscontaining, for example, polyoxyethylene-9-lauryl ether, glycocholateand deoxycholate, or may be oily solutions for administration in theform of nasal drops, or as a gel.

The formulations can be administered to human patients intherapeutically effective amounts (e.g., amounts which prevent,eliminate, or reduce a pathological condition) to provide therapy for adisease or condition. The preferred dosage of a nucleobase oligomer ofthe invention is likely to depend on such variables as the type andextent of the disorder, the overall health status of the particularpatient, the formulation of the compound excipients, and its route ofadministration.

As described above, if desired, treatment with a nucleobase oligomer ofthe invention may be combined with therapies for the treatment ofproliferative disease (e.g., radiotherapy, surgery, or chemotherapy).

For any of the methods of application described above, a nucleobaseoligomer of the invention is desirably administered intravenously or isapplied to the site of the needed apoptosis event (e.g., by injection).

Oligonucleotides and Other Nucleobase Oligomers

At least two types of oligonucleotides induce the cleavage of RNA byRNase H: polydeoxynucleotides with phosphodiester (PO) orphosphorothioate (PS) linkages. Although 2′-OMe-RNA sequences exhibit ahigh affinity for RNA targets, these sequences are not substrates forRNase H. A desirable oligonucleotide is one based on 2′-modifiedoligonucleotides containing oligodeoxynucleotide gaps with some or allinternucleotide linkages modified to phosphorothioates for nucleaseresistance. The presence of methylphosphonate modifications increasesthe affinity of the oligonucleotide for its target RNA and thus reducesthe IC₅₀. This modification also increases the nuclease resistance ofthe modified oligonucleotide. It is understood that the methods andreagents of the present invention may be used in conjunction with anytechnologies that may be developed, including covalently-closed multipleantisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303,2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS)oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCTPublication No. WO 00/61595), and large circular antisenseoligonucleotides (U.S. Patent Application Publication No. US2002/0168631 A1).

As is known in the art, a nucleoside is a nucleobase-sugar combination.The base portion of the nucleoside is normally a heterocyclic base. Thetwo most common classes of such heterocyclic bases are the purines andthe pyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric structure can be further joined to form a circular structure;open linear structures are generally preferred. Within theoligonucleotide structure, the phosphate groups are commonly referred toas forming the backbone of the oligonucleotide. The normal linkage orbackbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.

Specific examples of preferred nucleobase oligomers useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,nucleobase oligomers having modified backbones include those that retaina phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,modified oligonucleotides that do not have a phosphorus atom in theirinternucleoside backbone are also considered to be nucleobase oligomers.

Nucleobase oligomers that have modified oligonucleotide backbonesinclude, for example, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriest-ers, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity, wherein the adjacent pairs of nucleoside units are linked3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Representative United States patents thatteach the preparation of the above phosphorus-containing linkagesinclude, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of whichis herein incorporated by reference.

Nucleobase oligomers having modified oligonucleotide backbones that donot include a phosphorus atom therein have backbones that are formed byshort chain alkyl or cycloalkyl internucleoside linkages, mixedheteroatom and alkyl or cycloalkyl internucleoside linkages, or one ormore short chain heteroatomic or heterocyclic internucleoside linkages.These include those having morpholino linkages (formed in part from thesugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxideand sulfone backbones; formacetyl and thioformacetyl backbones;methylene formacetyl and thioformacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH.sub.2 component parts. RepresentativeUnited States patents that teach the preparation of the aboveoligonucleotides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; and 5,677,439, each of which is hereinincorporated by reference.

In other nucleobase oligomers, both the sugar and the internucleosidelinkage, i.e., the backbone, are replaced with novel groups. Thenucleobase units are maintained for hybridization with an OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, or uPAR nucleic acid molecule. One suchnucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). InPNA compounds, the sugar-backbone of an oligonucleotide is replaced withan amide containing backbone, in particular an aminoethylglycinebackbone. The nucleobases are retained and are bound directly orindirectly to aza nitrogen atoms of the amide portion of the backbone.Methods for making and using these nucleobase oligomers are described,for example, in “Peptide Nucleic Acids Protocols and Applications” Ed.P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999.Representative United States patents that teach the preparation of PNAsinclude, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331;and 5,719,262, each of which is herein incorporated by reference.Further teaching of PNA compounds can be found in Nielsen et al.,Science, 1991, 254, 1497-1500.

In particular embodiments of the invention, the nucleobase oligomershave phosphorothioate backbones and nucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— (knownas a methylene (methylimino) or MMI backbone), —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂—, and —O—N(CH₃)—CH₂—CH₂—. In other embodiments,the oligonucleotides have morpholino backbone structures described inU.S. Pat. No. 5,034,506.

Nucleobase oligomers may also contain one or more substituted sugarmoieties. Nucleobase oligomers comprise one of the following at the 2′position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- orN-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynylmay be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyland alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(n)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)nON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. Otherpreferred nucleobase oligomers include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl, or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of a nucleobase oligomer, or a group forimproving the pharmacodynamic properties of an nucleobase oligomer, andother substituents having similar properties. Preferred modificationsare 2′-O-methyl and 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl) or 2′-MOE). Another desirable modification is2′-dimethylaminooxyethoxy (i.e., O(CH₂)₂ON(CH₃)₂), also known as2′-DMAOE. Other modifications include, 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on an oligonucleotide or other nucleobaseoligomer, particularly the 3′ position of the sugar on the 3′ terminalnucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′terminal nucleotide. Nucleobase oligomers may also have sugar mimeticssuch as cyclobutyl moieties in place of the pentofuranosyl sugar.Representative United States patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, each of which is herein incorporated byreference in its entirety.

Nucleobase oligomers may also include nucleobase modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude the purine bases adenine (A) and guanine (G), and the pyrimidinebases thymine (T), cytosine (C) and uracil (U). Modified nucleobasesinclude other synthetic and natural nucleobases, such as5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine; 2-propyl and other alkyl derivatives of adenine andguanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouraciland cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine andthymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other5-substituted uracils and cytosines; 7-methylguanine and7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons,1990, those disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y.S., Chapter 15, Antisense Research and Applications, pages 289-302,Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of thesenucleobases are particularly useful for increasing the binding affinityof an antisense oligonucleotide of the invention. These include5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and aredesirable base substitutions, even more particularly when combined with2′-O-methoxyethyl or 2′-O-methyl sugar modifications. RepresentativeUnited States patents that teach the preparation of certain of the abovenoted modified nucleobases as well as other modified nucleobases includeU.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and5,750,692, each of which is herein incorporated by reference.

Another modification of a nucleobase oligomer of the invention involveschemically linking to the nucleobase oligomer one or more moieties orconjugates that enhance the activity, cellular distribution, or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., Proc.Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan etal., Bioorg. Med. Chem. Let, 4:1053-1060, 1994), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let.,3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol orundecyl residues (Saison-Behmoaras et al., EMBO J., 10:1111-1118, 1991;Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al.,Biochimie, 75:49-54, 1993), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids Res.,18:3777-3783, 1990), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett.,36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys.Acta, 1264:229-237, 1995), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 277:923-937, 1996. Representative United States patents thatteach the preparation of such nucleobase oligomer conjugates includeU.S. Pat. Nos. 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882;4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045;5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077;5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667;5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552;5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481;5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928;5,608,046; and 5,688,941, each of which is herein incorporated byreference.

The present invention also includes nucleobase oligomers that arechimeric compounds. “Chimeric” nucleobase oligomers are nucleobaseoligomers, particularly oligonucleotides, that contain two or morechemically distinct regions, each made up of at least one monomer unit,i.e., a nucleotide in the case of an oligonucleotide. These nucleobaseoligomers typically contain at least one region where the nucleobaseoligomer is modified to confer, upon the nucleobase oligomer, increasedresistance to nuclease degradation, increased cellular uptake, and/orincreased binding affinity for the target nucleic acid. An additionalregion of the nucleobase oligomer may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of nucleobase oligomerinhibition of gene expression. Consequently, comparable results canoften be obtained with shorter nucleobase oligomers when chimericnucleobase oligomers are used, compared to phosphorothioatedeoxyoligonucleotides hybridizing to the same target region.

Chimeric nucleobase oligomers of the invention may be formed ascomposite structures of two or more nucleobase oligomers as describedabove. Such nucleobase oligomers, when oligonucleotides, have also beenreferred to in the art as hybrids or gapmers. Representative UnitedStates patents that teach the preparation of such hybrid structuresinclude U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; and 5,700,922, each of which is herein incorporated byreference in its entirety.

The nucleobase oligomers used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

The nucleobase oligomers of the invention may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption assisting formulations includeU.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291;5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899;5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633;5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756,each of which is herein incorporated by reference.

Polynucleotide Therapy

Polynucleotide therapy is another therapeutic approach in which anucleic acid encoding a OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, oruPAR inhibitory nucleic acid molecule is introduced into cells. Thetransgene is delivered to cells in a form in which it can be taken upand expressed in an effective amount to inhibit neoplasia progression.

Transducing retroviral, adenoviral, or human immunodeficiency viral(HIV) vectors are used for somatic cell gene therapy because of theirhigh efficiency of infection and stable integration and expression (see,for example, Cayouette et al., Hum. Gene Ther., 8:423-430, 1997; Kido etal., Curr. Eye Res. 15:833-844, 1996; Bloomer et al., J. Virol.71:6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; Miyoshiet al., Proc. Natl. Acad. Sci. USA, 94:10319-10323, 1997). For example,OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR inhibitory nucleicacid molecules, or portions thereof, can be cloned into a retroviralvector and driven from its endogenous promoter, from the retroviral longterminal repeat, or from a promoter specific for the target cell type ofinterest (such as epithelial carcinoma cells). Other viral vectors thatcan be used include, but are not limited to, adenovirus,adeno-associated virus, vaccinia virus, bovine papilloma virus,vesicular stomatitus virus, or a herpes virus such as Epstein-BarrVirus.

Gene transfer can be achieved using non-viral means requiring infectionin vitro. This would include calcium phosphate, DEAE-dextran,electroporation, and protoplast fusion. Liposomes may also bepotentially beneficial for delivery of DNA into a cell. Although thesemethods are available, many of these are of lower efficiency.

Screening Methods

The invention provides methods for identifying agents useful for thetreatment or prevention of a neoplasia (e.g., prostate carcinoma).Screens for the identification of such agents employ prostate cancerstem cells identified according to the methods of the invention. The useof such cells, which express increased levels of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and/or uPAR is particularly advantageous for theidentification of agents that reduce the survival of this aggressivesubpopulation of prostate cancer cells. Agents identified as reducingthe survival, reducing the proliferation, or increasing cell death inOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and/or uPAR expressing cellare particularly useful.

Methods of observing changes in OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, or uPAR interactions and OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, or uPAR biological activity are exploited in high throughputassays for the purpose of identifying compounds that modulate OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR biological activity, e.g.,transciptional regulation or protein-nucleic acid interactions.Compounds that inhibit OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, oruPAR binding to a regulated gene, or that inhibit another OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, or uPAR biological activity (e.g., OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR's activity as atranscriptional activator or repressor), may be identified by suchassays. In addition, compounds that modulate the expression of a OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR polypeptide or nucleic acidmolecule whose expression is altered in a patient having a neoplasia maybe identified.

Any number of methods are available for carrying out screening assays toidentify new candidate compounds that decrease the expression of anOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR nucleic acidmolecule. In one example, candidate compounds are added at varyingconcentrations to the culture medium of cultured cells expressing one ofthe nucleic acid sequences of the invention. Gene expression is thenmeasured, for example, by microarray analysis, Northern blot analysis(Ausubel et al., supra), or RT-PCR, using any appropriate fragmentprepared from the nucleic acid molecule as a hybridization probe. Thelevel of gene expression in the presence of the candidate compound iscompared to the level measured in a control culture medium lacking thecandidate molecule. A compound which reduces the expression of a OOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR gene, or afunctional equivalent thereof, is considered useful in the invention;such a molecule may be used, for example, as a therapeutic to treat aneoplasia in a human patient.

In another example, the effect of candidate compounds may be measured atthe level of polypeptide production using the same general approach andstandard immunological techniques, such as Western blotting orimmunoprecipitation with an antibody specific for a polypeptide encodedby an OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR gene. Forexample, immunoassays may be used to detect or monitor the expression ofat least one of the polypeptides of the invention in an organism.Polyclonal or monoclonal antibodies (produced as described above) thatare capable of binding to such a polypeptide may be used in any standardimmunoassay format (e.g., ELISA, Western blot, or RIA assay) to measurethe level of the polypeptide. In some embodiments, a compound thatpromotes an increase in the expression or biological activity of thepolypeptide is considered particularly useful. Again, such a moleculemay be used, for example, as a therapeutic to delay, ameliorate, ortreat a neoplasia in a human patient.

In yet another working example, candidate compounds may be screened forthose that specifically bind to a polypeptide encoded by an OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR gene. The efficacy of sucha candidate compound is dependent upon its ability to interact with sucha polypeptide or a functional equivalent thereof. Such an interactioncan be readily assayed using any number of standard binding techniquesand functional assays (e.g., those described in Ausubel et al., supra).In one embodiment, a candidate compound may be tested in vitro for itsability to specifically bind a polypeptide of the invention. In anotherembodiment, a candidate compound is tested for its ability to inhibitthe biological activity of a polypeptide described herein, such as aOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR polypeptide. Thebiological activity of an OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8,or uPAR polypeptide may be assayed using any standard method, forexample, a matrigel cell invasion or cell migration assay.

In another working example, a nucleic acid described herein (e.g., anOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR nucleic acid) isexpressed as a transcriptional or translational fusion with a detectablereporter, and expressed in an isolated cell (e.g., mammalian) under thecontrol of a heterologous promoter, such as an inducible promoter. Thecell expressing the fusion protein is then contacted with a candidatecompound, and the expression of the detectable reporter in that cell iscompared to the expression of the detectable reporter in an untreatedcontrol cell. A candidate compound that alters the expression of thedetectable reporter is a compound that is useful for the treatment of aneoplasia. Preferably, the compound decreases the expression of thereporter.

In another example, a candidate compound that binds to a polypeptideencoded by an OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR genemay be identified using a chromatography-based technique. For example, arecombinant polypeptide of the invention may be purified by standardtechniques from cells engineered to express the polypeptide (e.g., thosedescribed above) and may be immobilized on a column. A solution ofcandidate compounds is then passed through the column, and a compoundspecific for the OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPARpolypeptide is identified on the basis of its ability to bind to thepolypeptide and be immobilized on the column. To isolate the compound,the column is washed to remove non-specifically bound molecules, and thecompound of interest is then released from the column and collected.Similar methods may be used to isolate a compound bound to a polypeptidemicroarray. Compounds isolated by this method (or any other appropriatemethod) may, if desired, be further purified (e.g., by high performanceliquid chromatography). In addition, these candidate compounds may betested for their ability to increase the activity of an OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, or uPAR polypeptide (e.g., as describedherein). Compounds isolated by this approach may also be used, forexample, as therapeutics to treat a neoplasia in a human patient.Compounds that are identified as binding to a polypeptide of theinvention with an affinity constant less than or equal to 10 mM areconsidered particularly useful in the invention. Alternatively, any invivo protein interaction detection system, for example, any two-hybridassay may be utilized.

Potential antagonists include organic molecules, peptides, peptidemimetics, polypeptides, nucleic acids, and antibodies that bind to anucleic acid sequence or polypeptide of the invention (e.g., an OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR polypeptide or nucleic acidmolecule).

Each of the DNA sequences listed herein may also be used in thediscovery and development of a therapeutic compound for the treatment ofneoplasia. The encoded protein, upon expression, can be used as a targetfor the screening of drugs. Additionally, the DNA sequences encoding theamino terminal regions of the encoded protein or Shine-Delgarno or othertranslation facilitating sequences of the respective mRNA can be used toconstruct sequences that promote the expression of the coding sequenceof interest. Such sequences may be isolated by standard techniques(Ausubel et al., supra).

Optionally, compounds identified in any of the above-described assaysmay be confirmed as useful in an assay for compounds that modulate thepropensity of a neoplasia to metastasize.

Small molecules of the invention preferably have a molecular weightbelow 2,000 daltons, more preferably between 300 and 1,000 daltons, andmost preferably between 400 and 700 daltons. It is preferred that thesesmall molecules are organic molecules.

Test Extracts and Agents

In general, agents that modulate OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, or uPAR expression, biological activity, or OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, or uPAR-dependent signaling are identifiedfrom large libraries of both natural products, synthetic (orsemi-synthetic) extracts or chemical libraries, according to methodsknown in the art. Preferably, these compounds decrease OCT3/4, Nanog,Sox2, c-Myc, Klf4, Keratin 8, or uPAR expression or biological activity.

Those skilled in the art will understand that the precise source of testextracts or compounds is not critical to the screening procedure(s) ofthe invention. Accordingly, virtually any number of chemical extracts orcompounds can be screened using the exemplary methods described herein.Examples of such extracts or compounds include, but are not limited to,plant-, fungal-, prokaryotic- or animal-based extracts, fermentationbroths, and synthetic compounds, as well as modifications of existingcompounds. Numerous methods are also available for generating random ordirected synthesis (e.g., semi-synthesis or total synthesis) of anynumber of chemical compounds, including, but not limited to,saccharide-, lipid-, peptide-, and nucleic acid-based compounds.Synthetic compound libraries are commercially available from, forexample, Brandon Associates (Merrimack, N.H.), Aldrich Chemical(Milwaukee, Wis.), and Talon Cheminformatics (Acton, Ont.)

Alternatively, libraries of natural compounds in the form of bacterial,fungal, plant, and animal extracts are commercially available from anumber of sources, including, but not limited to, Biotics (Sussex, UK),Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce,Fla.), and PharmaMar, U.S.A. (Cambridge, Mass.). In addition, naturaland synthetically produced libraries are produced, if desired, accordingto methods known in the art (e.g., by combinatorial chemistry methods orstandard extraction and fractionation methods). Furthermore, if desired,any library or compound may be readily modified using standard chemical,physical, or biochemical methods.

Combination Therapies

The present invention provides therapeutic compositions and methods forthe treatment of a neoplasia (e.g., prostate carcinoma), which may beused alone or in combination with any other cancer therapy known in theart. In particular, the invention provides agents (e.g., smallcompounds, polypeptides, polynucleotides) that inhibit the biologicalactivity of any one or more of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin8, and uPAR expression or biological activity. In one particularembodiment, the invention providesinhibitory nucleic acids that inhibitthe expression of any one or more of OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, and uPAR. Agents of the invention may be administered aloneor in any combination that is effective to treat prostate carcinoma. Ifdesired, agents of the invention are administered in combination withany other standard neoplasia therapy; such methods are known to theskilled artisan (e.g., Wadler et al., Cancer Res. 50:3473-86, 1990), andinclude, but are not limited to, chemotherapy, hormone therapy,immunotherapy (include, but are not limited to, immunotherapy that willspecifically target cancer stem cell transcription factors),radiotherapy, and any other therapeutic method used for the treatment ofneoplasia.

Kits

The invention provides kits for the treatment or prevention of neoplasia(e.g., prostate cancer). In one embodiment, the kit provides for thetreatment of prostate cancer that expresses Sox2, alone or incombination with one, two, three, four, five, or all of OCT3/4, Nanog,c-Myc, Klf4, Keratin 8, or uPAR. In one embodiment, the kit includes atherapeutic or prophylactic composition containing an effective amountof an inhibitory nucleic acid molecule that disrupts the expression ofan OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, or uPAR polynucleotideor polypeptide in unit dosage form. In another embodiment, the kitincludes a therapeutic or prophylactic composition containing aneffective amount of an inhibitory nucleic acid molecule that disruptsthe expression of OCT3/4, Nanog, and Sox2 polynucleotides orpolypeptides in unit dosage form. In yet another embodiment, the kitincludes a therapeutic or prophylactic composition containing aneffective amount of an inhibitory nucleic acid molecule that disruptsthe expression of an OCT3/4, Nanog, Sox2, and c-Myc polynucleotides orpolypeptides in unit dosage form. In some embodiments, the kit comprisesa sterile container which contains a therapeutic or prophylacticcellular composition; such containers can be boxes, ampoules, bottles,vials, tubes, bags, pouches, blister-packs, or other suitable containerforms known in the art. Such containers can be made of plastic, glass,laminated paper, metal foil, or other materials suitable for holdingmedicaments.

If desired an inhibitory nucleic acid molecule of the invention isprovided together with instructions for administering the inhibitorynucleic acid molecule to a subject having or at risk of developingprostate cancer. The instructions will generally include informationabout the use of the composition for the treatment or prevention ofprostate cancer. In other embodiments, the instructions include at leastone of the following: description of the therapeutic agent; dosageschedule and administration for treatment or prevention of ischemia orsymptoms thereof; precautions; warnings; indications;counter-indications; overdosage information; adverse reactions; animalpharmacology; clinical studies; and/or references. The instructions maybe printed directly on the container (when present), or as a labelapplied to the container, or as a separate sheet, pamphlet, card, orfolder supplied in or with the container.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are well within the purview of the skilled artisan.Such techniques are explained fully in the literature, such as,“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook,1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture”(Freshney, 1987); “Methods in Enzymology” “Handbook of ExperimentalImmunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells”(Miller and Calos, 1987); “Current Protocols in Molecular Biology”(Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques areapplicable to the production of the polynucleotides and polypeptides ofthe invention, and, as such, may be considered in making and practicingthe invention. Particularly useful techniques for particular embodimentswill be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the assay, screening, and therapeutic methods of theinvention, and are not intended to limit the scope of what the inventorsregard as their invention.

EXAMPLES Example 1 OCT 3/4, Nanog, Sox2, c-Myc, and Klf4 are Expressedin Cancer Stem Cells in Metastatic Prostate Cancer Cell Lines

Self-renewal is a unique property shared by both normal and cancer stemcells Cancer stem cells were examined for transcriptionalcharacteristics of embryonic stem cells. Embryonic stem celltranscription factors OCT3/4 and Nanog, which are responsible formaintaining self-renewal and pluripotency of undifferentiated embryonicstem cells, were used as markers to identify cancer stem cells inmetastatic prostate cancer cell lines. Reverse transcription PolymeraseChain Reaction (RT-PCR) was performed by standard methods to detect themRNA levels of these genes in DU145, LNCaP and PC3 cells. OCT3/4 andNanog expression were clearly detected at high levels in all of theprostate cancer cell lines (FIG. 1A). CD133 expression was also examinedbecause variant solid tumor stem cells have been isolated using CD133which is expressed on the cell surface (Collins et al., 2005; O'Brien etal., 2007; Singh et al., 2004). RT-PCR analysis showed that theexpression of CD133 was very low or absent in DU145, LNCaP and PC3 tumorcell lines. Western blot analysis also confirmed the expression ofOCT3/4 and Nanog proteins in the three prostate cancer cell lines (FIG.1B).

To examine whether OCT3/4 and Nanog were expressed as proteins byputative cancer stem cells in the tumor cell lines, DU145, LNCaP and PC3cultured cells were examined for OCT3/4 and Nanog expression byimmunofluorescence staining. A distinct population of cells displayedhigh expression of both OCT3/4 and Nanog by immunofluorescencemicroscopy (FIG. 1C). These cells were readily observed in all threecell lines, representing 5-10% of total cells. These results suggest theexistence of a small population of cancer stem cells in DU145, LNCaP andPC3 metastatic prostate tumor cell lines.

To investigate whether pluripotent stem cell reprogramming factors inaddition to OCT3/4 and Nanog were expressed by a subpopulation ofputative stem cell-like tumor cells in the prostate cancer cell lines,RT-PCR was used to evaluate the mRNA expression levels of the corepluripotent stem cell reprogramming factors SOX2, c-Myc, and Klf4 inDU145 and PC3 prostate cancer cell lines. In these studies, humanembryonic stem cell (hESC) line H9 was used as a reference. RT-PCRanalysis revealed that both prostate tumor cell lines expresseddetectable levels of mRNA for SOX2, c-Myc, and Klf4 as well as OCT3/4,Nanog, (FIG. 2A). Additionally, OCT 3/4 transcripts were confirmed andshown not to be those of the related pseudogenes, as assessed by themethod of Panagopoulos et. al. (2008. Genes Chromosomes Cancer47:521-529). Compared to the embryonic stem cell line H9, the prostatetumor cell lines displayed relatively low levels of OCT3/4, SOX2 andNanog. They did however express high levels of the oncogene c-Myc.Expression of Klf4, a context-dependent oncogene (15), was also elevatedin prostate tumor cells compared to embryonic stem cells.

In addition to gene expression analysis, the five reprogramming factorswere measured by Western blot analysis in the prostate tumor cell lines(FIG. 2B). Similar to the RT-PCR results for mRNA expression, DU145 andPC3 prostate tumor cells had lower expression of OCT3/4 protein andhigher expression of c-Myc and Klf4 proteins than embryonic stem (ES)cells. Western blot analysis of SOX2 and Nanog revealed similar orhigher levels of protein expression in the tumor cell lines compared tothe normal ES cells when compared with RT-PCR analysis of mRNAexpression. Taken together these data indicate that pluripotent stemcell reprogramming factors were activated in prostate cancer cells.

To investigate what population of putative stem cell-like tumor cellsexpress OCT3/4 and SOX2 transcription factors in DU145 and PC3 prostatecancer cell lines, DU145 and PC3 cultured cells were examined for OCT3/4and SOX2 expression by immunofluorescence staining. Immunofluorescentdouble staining for the two markers, showed that only a discretepopulation of tumor cells (˜5-10% of total cells) stained positive forboth OCT3/4 and SOX2 in the prostate tumor cell lines, (FIG. 2C) similarresults were observed with immunofluorescence staining of Nanog andOCT3/4. These results indicated the existence of a small population ofcancer stem cells in metastatic prostate tumor cell lines.

Example 2 Cancer Stem Cells Isolated from Metastatic Prostate Tumor CellLines Possessed High Tumorgenicity

To isolate cancer stem cells from the prostate cancer cell lines, cellsurface markers were screened by immunofluorescence microscopy forexpression in the metastatic prostate cancer stem cells. DU145, LNCaPand PC3 cell lines were analyzed with an antibody panel of selected cellsurface associated proteins in pluripotent stem cells and cancer stemcells, including CD9, E-cadherin, PODXL, SSEA1, SSEA4, CD24, and CD133.The tumor cell lines were also analyzed for the stem cell marker OCT3/4to detect the cancer stem cells. Of the cell surface markers screened byco-immunofluorescence among the DU145, LNCaP and PC3 cell lines,E-cadherin showed high levels of expression in cells also expressingOCT3/4 (FIG. 3A). The expression levels of all the other markers testeddid not correlate with OCT3/4 expression among the three prostate cancercell lines. These results were further confirmed with flowcytometry-based analysis which showed that PC3 stem cells could besorted from non-stem cancer cells (FIG. 3B). Based on these results,stem cell populations were separated from the three prostate cancer celllines based on their E-cadherin expression profile using FACS analysis(Becton Dickinson MoFlo cell sorter) (FIG. 3C). In addition to the FACSanalysis, the cell sorting method was further validated with RT-PCRassays using primers specific for E-cadherin, Nanog and OCT3/4. FACSsorting resulted in an enriched cancer cell population exhibiting highmRNA levels for the stem cell transcription factors Nanog and OCT3/4 asmeasured by RT-PCR (FIG. 3D).

To assess the function of stem cell-like tumor cells that expresspluripotent stem cell transcription factors in prostate cancer,potential cell surface markers of prostate stem cell-like tumor cellsfor cell sorting were screened in DU145 and PC3. The key stem cellregulator OCT3/4 was used as a marker to identify tumor cells withhighly elevated stem-cell reprogramming factors. Once identified, thesecells were then co-stained with a panel of cell surface antibodiesselected on the basis of their association with pluripotent stem cellsand cancer stem cells. These included CD44, ESA, and Integrin-α2β1 inaddition to CD9, E-cadherin, PODXL, SSEA1, SSEA4 CD24, CD133, describedabove. The results showed that OCT3/4 nuclear positive cells wereexclusively located in colonies that displayed classic morphology asmalignant holoclones comprised of groups of tightly packed smaller tumorcells (Li et al., 2008. Cancer Res. 68:1820-1825.). Like holoclones fromother carcinoma-derived cell lines, the holoclones from prostate tumorcell lines exhibited high expression of the epithelial marker E-cadherin(Locke et al., 2005. Cancer Res. 65:8944-8950). Indeed, most of OCT3/4positive cells in the prostate tumor cell lines had high surfaceexpression of E-cadherin (FIG. 4A). E-cadherin low or negative coloniescontained few OCT3/4 positive tumors cells. Interestingly, PC3, which isknown to have reduced surface E-cadherin expression due to the deletionof α-catenin gene, also displayed co-localized nuclear OCT3/4 stainingwith cytoplasmic E-cadherin staining (Morton et al., 1993. Cancer Res.53:3585-3590). All other surface markers evaluated were detected atvarying expression levels among the prostate cancer cell lines but thesedid not co-localize with OCT3/4 staining.

Importantly, E-cadherin positive cells exhibited not only OCT3/4positive staining but also high expression of CD44 and Integrin-α2β1 asmeasured by flow cytometry analysis (FIGS. 4B and 4C). In both DU145 andPC3 cells expression of CD44 is exceedingly high (˜90% DU145, ˜100%PC3), making it difficult to isolate the stem cell population using onlythis marker. Investigators studying prostate cancer cell lines thereforetypically have turned to using CD44 in combination with other markersincluding CD24, CD133, and Integrin-α2β1.

Putative stem cell-like populations were isolated from both prostatecancer cell lines by flow cytometry on the basis of the E-cadherinexpression profiles. In these studies 17% of DU145 cells and 5.5% of PC3cells were found to be positive for E-cadherin based on the isotypecontrol (FIGS. 4D and 4E). Highly purified sub-populations of cells wereobtained by isolating the top 5-10% of the cells highly expressingE-cadherin or the bottom 5-10% without E-cadherin expression. To confirmthe enrichment of stem cell-like tumor cells after cell sorting, thegene expression of pluripotent stem cell reprogramming factors in theE-cadherin⁺ and E-cadherin⁻ populations was examined at the mRNA level(FIG. 4F). The data showed that compared to E-cadherin⁻ cells, only theE-cadherin⁺ cells expressed all five essential pluripotent stem cellreprogramming factors: OCT3/4, SOX2, Nanog, c-Myc and Klf4. Thus,E-cadherin, which showed distinguishable expression in the two celllines (˜17% DU145 and ˜5.5% PC3), was utilized as a solitary, reliableand discrete marker for isolating the stem-like cell population fromprostate cancer cell lines. These results indicated that E-cadherin canserve as a distinct surface marker to isolate prostate tumor initiatingcells in these two cell lines and does not require combinatorialstaining.

Self-renewal, proliferation, and differentiation are hallmarks of stemcells. To test the clonogenic capacity of isolated stem cells, theprostate tumor stem cells isolated by FACS analysis were cultured insemisolid medium of soft agar for 2-3 weeks until colonies werewell-formed. For each cell line, tumor stem cells formed larger and moreclones than non-stem tumor cells (P<0.01) (FIG. 5A). This difference wasnot due to the adhesion properties conferred by E-cadherin in thepositive cells, as approximately equal numbers of E-cadherin⁺ andE-cadherin⁻ cells attached upon initial plating. Because both normal andneoplastic prostate stem cells from epithelial origin can be expandedunder spheroid culture conditions, sorted E-cadherin⁺ and E-cadherin⁻DU145 tumor cells were cultured in serum-free medium containing EGF andbFGF under low-attachment conditions in order to favor the proliferationof undifferentiated cells. The results showed that only the E-cadherin+cells had the ability to form prostate spheroids (FIG. 5B). Western blotanalysis of the spheroid culture generated from these E-cadherin+ cellsfurther revealed elevated levels of the stem cell reprogramming factorsOCT3/4 and SOX2 as compared to the unsorted parental DU145 cell line(FIG. 5B).

The proliferative capability of the cancer stem cells was alsodemonstrated (FIG. 5C). Stem and non-stem cells sorted from prostatecancer cell lines were plated and observed. To confirm that prostatestem cell-like tumor cells possess self-renewal capacity, E-cadherin⁺and E-cadherin⁻ DU145 cells were evaluated by immunofluorescent analysisusing E-cadherin and β-catenin antibodies. The stem cells showed ahigher rate of proliferation compared to non-stem cells which displayedlittle or slow proliferation. Cells grown from the cancer stem cells,which express E-Cadherin, could also differentiate into two populations(E-cad positive and E-cad negative) as observed by immunofluorescence(FIG. 5C). After 3 days in culture, both populations were positive forβ-catenin, but the E-cadherin⁻ cells proliferated slowly and remainednegative for E-cadherin. In contrast, the E-cadherin⁺ cell populationwas not only highly proliferative but also produced both E-cadherin⁺ andE-cadherin⁻ subpopulations, suggesting that asymmetrical divisionoccurred during culture and that the E-cadherin⁺ cell population wasenriched with stem cells. A transwell assay was used to observe theinvasiveness of cells, in which cells are observed for the ability tomigrate from one layer to another through holes in the plates. In thetranswell assay, prostate cancer stem cells displayed more migration,thus more invasiveness, compared to non-stem cancer cell (FIG. 5D).

The cell adhesion molecule E-cadherin, one classic marker for epithelialcells, has previously been shown to play an important role maintainingthe undifferentiated stage of ES cancer stem cells (Eastham et al. 2007.Cancer Res. 67:11254-11262) and to be down-regulated through theepithelial to mesenchymal transition (EMT) during ES celldifferentiation. Interestingly, carcinoma cells utilize a similarmechanism to obtain migratory and invasive capability (Theiry, 2002.Epithelial-mesenchymal transitions in tumour progression. Nat. Rev.Cancer 2:442-454). Although, down regulation of E-cadherin has beenthought to be correlated with highly invasive tumors and poor prognosisin prostate cancer, several studies fail to support this notion (Rubinet al., 2001. Hum. Pathol. 32:690-697; Saha et al., 2008. Prostate68:78-84; Tsukino et al., 2004. Urol. Int. 72:203-207; Yates et al.,2007. Co-culturing human prostate carcinoma cells with hepatocytes leadsto increased expression of E-cadherin. Br. J. Cancer 96:1246-1252). Forexample, high expression of E-cadherin was observed in prostatecarcinoma bone metastases suggesting the transient nature of EMT (Rubinet al., 2001. Hum. Pathol. 32:690-697; Saha et al., 2008 Prostate68:78-84; Tsukino et al., 2004. Urol. Int 72:203-207). Other studiessuggest that malignant prostate tumor cells, including the α-catenindeleted PC3 cells, up-regulate E-cadherin upon contact with host cellsat the site of metastasis such as liver (Yates et al., 2007. Br. J.Cancer 96:1246-1252) and that the TGF-β induced EMT depletes the stemcell enriched “side population” in breast cancer cells (Yin et al.,2008. Cancer Res. 68:800-807.). Taken together these data suggest thattumor cells only transiently down-regulate E-cadherin for invasion andre-expression of E-cadherin occurs after metastatic seeding (Chafer etal., 2006. Cancer Res. 66:11271-11278). The present findings areconsistent with the above evidence that the E-cadherin+ cells inprostate tumor cell lines may have incomplete EMT and represent a stemcell-like subpopulation. The complete EMT cells, the E-cadherin− cells,may eventually lose self-renewal and proliferative capacity. Similar tothe present findings, others have also reported E-cadherin to be highlyexpressed among stem cell-enriched holoclonal carcinoma cells (Locke etal., 2005. Cancer Res. 65:8944-8950) and tumor spheres (Lang et al.,2001. Br. J. Cancer 85:590-599).

To evaluate the tumorigenic potential of prostate cancer stem cells invivo, tumor development experiments were performed in male SCID miceusing FACS-sorted PC3 and DU145 cancer stem cells. Stem-like ornon-stem-like populations (PC3 and DU145) were injected subcutaneouslyinto the mice. Because the two cell lines used have different geneticbackgrounds which affect tumor formation, different doses were used inthe experiments (1×10³ PC3 cells/mouse, FIG. 6B; and 1×10⁵ DU145cells/mouse, FIG. 6C). Animals were monitored; and tumor sizes weremeasured. Mice that were injected with cancer stem cells all developedprostate cancer solid tumors within 30 days after cell inoculation anddied within 80 days after cell inoculation (FIGS. 6A-6C). In contrast,mice receiving non-stem cancer cells did not develop tumors during 80days of observation. These results suggest that the prostate cancer stemcells, isolated and characterized under the conditions described here,possessed higher clonogenic and tumorigenic capacity than non-stemprostate cancer cells and were capable of initiating tumors.

Example 3 Metastatic Prostate Tumor-Initiating Cells Expressed FiveTranscription Factors Important for the Induction of Pluripotent StemCells from Somatic Cells

Studies have shown that the embryonic genes, such as OCT3/4 and Nanog,may function in the self-renewal of pluripotent stem cells Furthermore,a recent study by Yamanaka's group showed that c-Myc, Klf4, Sox2 andOCT3/4 may function in the induction of pluripotent stem cells fromsomatic cells (Takahashi and Yamanaka, 2006). To determine whetherc-Myc, Klf4, Nanog, Sox2 and OCT3/4 play an important role in cancer,the expression of these genes was analyzed by RT-PCR in prostatetumor-initiating cells and non-stem tumor cells purified from the DU145,LNCaP and PC3 cell lines. Increased mRNA levels of Klf4, Nanog, OCT3/4and Sox2 were observed in the tumor-initiating cells compared tonon-stem tumor cells (FIG. 7A). Klf4 and Sox2 expression was enhancedwithin the tumor-initiating cell populations, compared to non-stemcells, among all three types of metastatic prostate cancer cell lines.High mRNA levels of c-Myc were observed in both cell populations for allprostate cancer cell lines. Western-blotting analysis confirmed similarprotein expression patterns for c-Myc, Klf4, Nanog, OCT3/4 and Sox2(FIG. 7B).

Example 4 c-Myc, Klf4, Nanog, OCT3/4 and Sox2 are Expressed In HumanProstate Cancer Tumor Tissue

Because the results indicated that a population of stem cells werepresent in prostate cancer cell lines, human prostate cancer tumortissue was also examined for the presence of prostate cancer stem cells.Without being bound to any particular theory, prostate neoplasia couldarise from the proliferation of prostate cancer stem cells, which arisefrom the mutation of normal stem cells in the prostate or thede-differentiation of differentiated cells in the prostate (FIG. 8A).Prostate cancer stem cells from tumors would be expected to express theOCT3/4, Sox2, c-Myc, and Nanog markers observed in the prostate celllines. RT-PCR analysis of four separate tumor tissue samples unenrichedfor stem cells demonstrated a similar expression profile for the OCT3/4,Sox2, c-Myc, and Nanog markers compared to the isolated stem cells fromthe prostate cancer cell lines (FIG. 8B). Prostate specific antigen(PSA) and androgen receptor (AR), prostate tissue-specific markers werehighly expressed in prostate tumor tissue. As the isolated prostate stemcells are undifferentiated, they were not expected to express PSA. Insitu immunohistochemical analysis on prostate tumor tissue revealedcells with high expression of OCT 3/4 and SOX2 in a small population ofcells, which were not observed in normal prostate tissue (FIGS. 8C and8D). These results show that c-Myc, Klf4, Nanog, OCT3/4 and Sox2 markerscan be used to identify prostate cancer stem cells and that prostatecancer stem cells are present in prostate tumors.

The expression of pluripotent stem cell reprogramming factors in humanprostate carcinoma was further examined by RT-PCR analysis of tumortissue samples from 55 prostate cancer patients and compared to a poolednormal prostate tissue sample from 32 Caucasian males. The hESC line H9served as a positive control. OCT3/4, SOX2, Nanog, c-Myc, and Klf4 mRNAtranscripts for were elevated in more than 50% of the prostate cancersamples compared to the normal prostate tissue pool (FIGS. 9A-9E).Densitometric analysis of mRNA transcripts revealed up to 6-foldactivation (after standardizing to normal tissue) of these pluripotentstem cell reprogramming genes in prostate cancer samples compared to thenormal prostate tissue pool. The expression pattern of these stem cellreprogramming genes was heterogeneous among patients.

Previously, a primary prostate stem cell-like line was isolated frommalignant human tumors that exhibit a stem cell-like phenotype in aneurosphere culture system, and established in vitro under conditionsthat exploit anchorage independence, serum starvation, and in thepresence of pleiotropic growth factors epithelial growth factor (EGF)and basic fibroblast growth factor (bFGF) (Gibbs et al., 2005. Neoplasia7:967-976). Total RNA from the primary prostate stem cell-like line wasextracted for RT-PCR analysis. These primary prostate cancer cells(prostate tumor sphere cells (PS, crosshatch)) were found to haveelevated expression of OCT3/4, SOX2, Nanog, c-Myc and Klf4 consistentwith a stem cell-like phenotype (FIGS. 9A-9E).

The correlation among the five transcription factors was analyzed usinga Newman Keuls multiple comparison test. Both the prostate sphereculture and the ES cell culture were statistically different from thenormal tissue pool and also from each other, with two exceptions. In theSOX2 analysis, the prostate sphere culture was not statisticallydifferent from the ES cell culture. In the Klf4 analysis, the ES cellculture was not statistically different from the normal tissue pool.Most importantly, in the 55 prostate tissue samples Spearman analysis onall possible combinations of transcription factors demonstrated thatsignificance was reached between OCT3/4 and SOX2 (Spearman correlationcoefficient of 0.4730, p<0.0001) suggesting a possible functional linkbetween OCT3/4 and SOX2 in prostate cancer (FIG. 9F).

To identify a stem cell-like subpopulation in primary prostate tumortissue, immunohistochemical staining (FIG. 10A) and analysis of the keystem cell regulators OCT3/4 and SOX2 was performed. The stainingintensity of these factors was evaluated using a tissue microarraycomprised of two core tissue samples from each of 35 localized prostatetumors (Gleason scores from 5 to 8) as well as 5 benign prostatehyperplasia (BPH) tissues. Nuclear OCT3/4 and SOX2 staining was observedin 76 and 81% of prostate tumor tissues respectively, but not in the BPHsamples. The extent of nuclear positive staining in the prostate tumorsamples varied widely.

Consequently, the data were stratified into 4 staining categories:negative, low (<5%), intermediate (5-25%), or high (26-50%); none of thesamples showed more than 50% nuclear staining. Representative patternsof nuclear staining are shown in FIG. 10A. The number of OCT3/4 or SOX2expressing cells was significantly lower in the normal prostate and BPHsamples as compared to the prostate tumor tissues (FIGS. 10B and 10C).Further, in the prostate tumor tissues samples, increasing numbers ofOCT3/4 and SOX2 expressing cells were evident with increasing Gleasonscores, suggesting that these cells play a role during prostate cancerprogression.

Defined stem cell transcription factors OCT3/4, SOX2, Nanog, c-Myc andKlf4 have been recently reported for reprogramming pluripotent stemcells from differentiated somatic cells (Takahashi and Yamanaka, 2006.Cell 126:663-676; Okita et al., 2007. Nature 448:313-317; Wernig et al.,2007. Nature 448:318-324.). Similar to tumor cells, the transformed orso-called induced pluripotent stem cells (iPS) are immortal, proliferaterapidly and form tumors in immune-deficient mice. As a group, these fivetranscription factors clearly demonstrate their putative role intransforming adult somatic cells. In the results described herein, thesefive stem cell transcription factors were expressed not only in thepluripotent stem cells, but also in prostate tumor-initiating cells.Without being bound to any particular theory, the existence of these EScell genes in both tumor-initiating cells and iPS cells suggest that theexpression and distribution of these five factors might be important fordetermining the fate of these adult stem cell-like cells during theevolution of a normal to a cancerous stem cell.

Example 5 Prostate Cancer Stem Cells are Resistant to ConventionalCancer Treatments and are Immune Privileged or Immunosuppressive

Prostate cancer stem cells from metastatic prostate cancer cell lineswere examined for their sensitivity to conventional cancer treatments(e.g., radiation and chemotherapy). Irradiation performed on metastaticprostate cancer stem cell line resulted in the increased detection ofSox2, Oct 3/4 and Nanog expression, possibly due to the enrichment ofprostate cancer stem cells with increasing radiation dose (FIG. 11A).When surviving fractions were quantified, prostate cancer stem cellsdemonstrated more resistance to radiation than non-stem prostate cancerstem cells (FIG. 11B). Metastatic prostate stem cell lines were alsotreated with Docetaxel, a frontline treatment for drug-resistant cancercells. Treatment with Docetaxel also resulted in the increased detectionof Sox2, Oct 3/4 and Nanog expression with increasing dose (FIG. 12A).Prostate cancer stem cells showed more cell viability compared tonon-stem prostate cancer cells, when both cell types were exposed toDocetaxel (FIG. 12B). These results showed that prostate cancer stemcells were resistant to conventional cancer treatments.

Because the cancer stem cells were relatively refractory to conventionaltherapies, which are unlikely to be curative and relapses would beexpected from prostate cancer stem cells. Prostate cancer stem cellswere also studied for treatment using targeted, active immunotherapy(Schuler et al., Curr Opin Immunol. 2003 April; 15(2):138-47, 2003),which employs the cancer stem cell-specific cytotoxic T cells patientsown immune system. To explore this possibility, MHC class I antigenpresenting pathway in the enriched prostate tumor-initiating cells werescreened by RT-PCR analysis. Various defects in expression were observedin stem cells from all three prostate cancer cell lines: DU145tumor-initiating stem cells have downregulated TAP1 expression; LNCaPtumor-initiating stem cells have low expression of LMP7 and TAP2; andPC3 tumor-initiating cells have little or not expression of LMP7 andTAP2 (FIG. 13A). Thus, the data suggests that genetic defects in theantigen presenting machinery of prostate tumor-initiating stem cells mayinhibit antigen presentation in prostate stem cells. These resultssuggest that prostate cancer stem cells may evade the immune system viadefects in the MHC class I antigen presenation pathway.

The ability of T-cells to identify prostate cancer stem cells was alsoanalyzed by IFN-γ ELISPOT. In the IFN-γ ELISPOT assay, T-cells are mixedwith sample cells and the T-cells secrete IFN-γ upon recognition oftumor cells, which is indicated by the detection of IFN-γ within acolony of tumor cells. LNCaP prostate cancer stem cells, which do notexpress CD44, were used in the IFN-γ ELISPOT assay (FIG. 13B). LNCaPprostate cancer stem cells showed low levels of detection by T-cells,although still higher than when MHC antigens were completely blocked byHLA antibody (FIG. 13C). When cancer stem cells were exposed toE-cadherin blocking antibody, there was a 4-fold increase in therecognition. The difference in the indicates that are able to avoidT-cell detection and are immune privileged or immunosuppressive.

Example 6 Disruption of the Stem Cell Transcriptional Balance Resultedin Cell Death in the Metastatic Prostate Tumor-Initiating Cells

To explore the function of c-Myc, Klf4, Nanog, OCT3/4 and Sox2 stem celltranscription factors, siRNAs specific for these targets were used toinhibit their gene expression. siRNAs specific for targeting c-Myc,Klf4, Nanog, OCT3/4 and Sox2 successfully reduced the expression of theselected genes, as confirmed by RT-PCR analysis, showing thedown-regulation of the corresponding genes in the tumor-initiating cells(FIG. 14A).

To examine the stem cell transcriptional balance in the tumor-initiatingcells from the metastatic prostate cancer cell lines, tumor-initiatingcells and non-stem tumor cells that were purified from DU145, LNCaP andPC3 cells were treated with c-Myc, Klf4, Nanog, OCT3/4 and Sox2 siRNAsseparately. Cell death was analyzed using a flow cytometric basedannexin V/propidium iodide (PI) binding assay (Lecoeur et al., 2001Cytometry. 2001 May 1; 44(1):65-72.). siRNAs targeting c-Myc, Klf4,Nanog, OCT3/4 or Sox2 induced cell death in a large percentage oftumor-initiating cells from DU145, LNCaP and PC3 cells (P<0.05, comparedwith control siRNA) (FIG. 14B). Specifically, after treatment with siRNAfor each of the five genes, numbers of live cells (annexin⁻/PI⁻) in thetumor-initiating cell population were significantly reduced. Incontrast, annexin V/PI double staining indicated a very low level ofcell death in the non-tumor-initiating cells. Each siRNA for c-Myc,Klf4, OCT3/4 or Sox2 induced more than 50% cell death in all threeprostate tumor-initiating cell types, especially in the LNCaP cell linewhere cell death was observed to be more than 70%. The siRNA for Nanoghad less impact on cell death when compared to the other four factors.Disruption of the stem cell transcriptional balance induced moreannexin⁻/PI⁺ cells in tumor-initiating cells from the DU145 and PC3lines than the cells from the LNCaP line which had a high percentage ofannexin cells. These results demonstrate that the transcriptionalbalance of c-Myc, Klf4, Nanog, OCT3/4 and Sox2 is important to thesurvival of tumor-initiating cells derived from these well-knownmetastatic prostate tumor lines.

The identification of stem cell-like tumor-initiating cells in prostatecancer models offers tremendous utility in further defining the natureand therapeutic vulnerability of putative prostate cancer stem cells.Data presented here reveal the importance of maintaining transcriptionalbalance for the survival of tumor-initiating cells. Interruption of thisbalance, for example, via the change of a single transcription factor,resulted in inhibition of tumor growth in vivo. These findings may havesignificant implications for identifying new strategies for cancertreatment (Dean et al., 2005. Nat. Rev. Cancer 5:275-284; Diehn et al.,2006. J. Natl. Cancer Inst. 98:1755-1757; Dingli et al., 2006. StemCells 24:2603-2610).

Example 7 Inhibition of In Vivo Tumorigenicity Using Oct3/4 or Sox2Short Hairpin RNAs (shRNA)

To gain further insights into the importance of stem-cell transcriptionfactors in tumorigenicity, DU145 prostate cancer cells were infectedwith plasmids encoding shRNAs targeting OCT3/4 or Sox2 or with shRNAcontrol plasmids. The effect of inhibiting OCT3/4 and Sox2 in prostatecancer stem cells was examined in the SCID mouse model oftumorigenicity. Prostate cancer stem cells were pre-treated with siRNAsor shRNAs, before being subcutaneously injected into mice. Both OCT3/4and SOX2 shRNA sequences individually dramatically reduced theexpression of their respective protein (FIG. 15A). Equal numbers ofOCT3/4 shRNA, Sox2 shRNA or control shRNA-transfected DU145 cells thenwere inoculated into SCID mice and tumor growth was monitored. Miceinjected with prostate cancer stem cells pre-treated with either OCT3/4shRNA or Sox2 shRNA failed to develop detectable tumors over anobservation period of 10 weeks (FIGS. 15B and 15C). In contrast, cellsinfected with control shRNA plasmids developed detectable tumor growth(4 out of 5 mice) within 3 weeks of cell inoculation. Mice receivingprostate cancer stem cells pre-treated with OCT3/4 and Sox2 siRNAs hadsmaller tumors than the control treated prostate cancer stem cells (FIG.15D). When used in combination, siRNAs and shRNAs for OCT3/4 and Sox2would have a greater effect on the reduction of tumor size. The resultsshow that the transcriptional balance of OCT3/4 and Sox2 is important tothe tumorigenicity of prostate stem cells.

Example 8 Nanog, OCT3/4 and Sox2 are Detected in Peripheral Blood ofProstate Cancer Patients

Peripheral blood samples or peripheral blood mononuclear cells (PBMC)from prostate cancer patients were analyzed for the presence ofpluripotent stem cell reprogramming factors, including OCT3/4, Sox2,Nanog, c-Myc, Klf4, Keratin 8, and uPAR. Samples from healthyindividuals (N1-N3) and prostate cancer patients (P1-P9) were analyzedby RT-PCR analysis. Information regarding the samples analyzed by RT-PCRanalysis, including that from the healthy donors (3) and prostate cancerpatients (9) is provided in Table 1.

TABLE 1 Information for Normal and Patient Samples. Samples Numbers Celltype Source Information Normal 3 PBMC Heiser N1, N2, N3 Lab Patient 9PBMC AllCells Normal peripheral blood mononuclear cells RNA, pooled froma minimum of 10 donors Duke P1: K./CH7279-52301/PBMC-60601 P2:Y.F./C69984-42601/PBMC-60601 P3: D.B./20601/PBMC-21601 P4: T.M.S.PRE/TMS-16-TRT/060303 DRY P5: screen#2/FSH-19-LMP/092403 P6:LMP-RNB-14/DOB 0408471071405 DRY P7: J.L.A./JLA-09-TRT/042302 DRY P8:J.D.S./JDS-11-TERT/061102 DRY P9: R.N.R POST/RNR-04-TRT/051302 DRY

RT-PCR was used to analyze the samples from peripheral blood (FIG. 16A)or peripheral blood mononuclear cells (PBMC) (FIG. 16B). Human embryonicstem cells were used as a positive control for the expression of themRNA for the pluripotent stem cell reprogramming factors andβ-microglobulin levels served as an internal control. Increased levelsof OCT3/4, SOX2, and Nanog mRNA were detected in peripheral bloodsamples of prostate cancer patients compared to samples from healthydonors with respect to β-microglobulin levels (FIG. 16A). Increasedlevels of OCT3/4, SOX2, and Nanog mRNA were also detected in peripheralblood samples of prostate cancer patients compared to a mRNA referencesample from normal peripheral blood mononuclear cells (FIG. 16B).Semi-quantitative RT-PCR analysis was applied to PBMC from 9 prostatecancer patients and compared to pooled normal PBMC from 13 normalhealthy donors (10 males and additional 3 individual normal healthydonors) (FIG. 16C). Band intensities were calculated using commerciallyavailable quantitation software (AlphaEase software, AlphaInnotech).Transcript levels for each case were normalized to β-microglobulinexpression and are represented in the graph as relative unitsstandardized to the averaged normal expression in PBMC. Significantlyincreased levels of OCT3/4, SOX2, and Nanog were detected in peripheralblood mononuclear cells (PBMC) of prostate cancer patients compared tonormal peripheral blood mononuclear cells. In particular, high transciptlevels of SOX2 correlated with the likelihood of a prostate cancerpatient to die from disease. Thus, SOX2 could be used as a marker topredict patient outcome in prostate cancer.

RT-PCR analysis was used to assess the expression level of OCT3/4, SOX2,Nanog, c-Myc, Klf4, and β-microglobulin (internal control) in peripheralblood mononuclear cells (PBMC) of prostate cancer patients undergoingvaccination (FIG. 17). Patients in the study were immunized with sixweekly doses of human telomerase reverse transcriptase hTERT(16-TERT),six weekly doses of lysosome-associated membrane protein-1 (LAMP)hTERT(14-LAMP; 19-LAMP), or three weekly cell doses of hTERT-(4-TERT;9-TERT; 11-TERT).

These studies show that increased OCT3/4, SOX2, and Nanog transcriptlevels were detected in peripheral blood samples in prostate cancerpatients, and that these levels indicate the presence of prostate cancerin this population of individuals.

Example 9 Prostate Cancer Cells Isolated as E-Cad⁺ are Invasive andExpress High Levels of Nanog, OCT3/4, Klf4 and Sox2

DU145 and PC3 prostate cancer cell lines were sorted by FACS accordingto E-cadherin surface expression (FIGS. 18A and 18B). After sorting,cells were cultured under sphere-forming conditions for 2-3 weeks, andwere confirmed to express E-cadherin and the embryonic stem cell markersSOX2, OCT3/4, Nanog, and Klf4 (FIG. 18C). The E-cad⁺ and E-cad⁻subpopulations were examined for their invasive abilities. The highlypurified E-cad⁺ cells isolated from both cell lines efficiently invadedthrough Matrigel whereas E-cad⁻ cells were only minimally invasive (FIG.19A; FIG. 18D).

Invaded E-cad⁺ and E-cad⁻ subpopulations were cultured under adherent ornon-adherent spheroid culture conditions. After 3 days of culture underadherent conditions, E-cad⁺ cells efficiently proliferated (FIG. 19B)and exhibited a holoclone morphology (FIG. 19C); stem cellcharacteristics previously demonstrated in DU145 and PC3 cells (Bae etal., J Urol 183: 2045-2053, 2010). The holoclone cells exhibited highlevels of E-cadherin expression (FIG. 19C). Furthermore, invaded DU145and PC3 E-cad⁺ cells that were cultured under non-adherent conditionsefficiently formed spheroids, which expressed E-cadherin and theprostate cancer stem cell marker CD44 at high levels (FIGS. 19D-19F). Incontrast, E-cad⁻ cells were unable to form spheroids (FIGS. 19D-19F).These results indicated that E-cad⁺ cells retain their ability to act ascancer stem cells and are primarily responsible for basement membraneinvasion.

According to the current E-cadherin literature, it was expected thatE-cad⁺ cells would invade poorly and that E-cad⁻ cells would be highlyinvasive, because the E-cad⁻ population is functionally equivalent toE-cadherin-knockdown cells, which have been demonstrated to be highlyinvasive. However, the robust invasion of E-cad⁺ cells was repeatedlyobserved. To analyze the mechanism by which E-cad⁺ cells invaded, thecourse of E-cadherin expression during the invasion process wasexamined. Initially, an invasion assay was used to examine E-cadherinexpression in invaded cells in the bottom chamber and the non-invadedcells of the top chamber at the end of a 24 h invasion period.Surprisingly, the E-cad⁺ cells residing in the top chamber (FIG. 19G,panel a; FIG. 19H, panel a) exhibited decreased E-cadherin expressioncompared to the invaded E-cad⁺ cells on the underside of the membrane(FIG. 19G, panel c; FIG. 19H, panel c). In contrast, E-cad⁻ cellsexhibited no E-cad staining either before (FIG. 19G, panel b; FIG. 19H,panel b) or after invasion (FIG. 19G, panel d; FIG. 19H, panel d).

To confirm that altered E-cadherin expression was concomitant withprogressive invasion, sorted E-cad⁺ DU145 and PC3 cells residing in thetop (FIG. 19I) or bottom (FIG. 19J) chamber were stained with anE-cadherin antibody 1 or 4 hr, respectively, after initiating theinvasion experiment. The results demonstrated that while the majority ofE-cad⁺ cells in the top invasion chamber exhibited extensive and largelyuniform E-cadherin expression at the beginning of the invasion process(FIG. 19I), cells that invaded through the Matrigel and emerged at thebottom of the chamber 4 hr later were completely void of E-cadherin(FIG. 19J). These findings showed a potential for the E-cad⁺ cells toeffectively modulate their E-cadherin expression during Matrigelinvasion. In contrast, E-cad⁻ cells which lacked E-cadherin at the startof invasion (FIG. 19G, panel b; FIG. 19H, panel b) did not expressE-cadherin post-invasion (FIG. 19G, panel d; FIG. 19H, panel d). Greaternumbers of parental DU145 and PC3 cells expressing E-cadherin at the endof a 24 hr invasion period (FIG. 18E) were found on the bottom of themembrane compared to those remaining on the top. Taken together, theseobservations suggest that E-cad⁺ cells capable of invading (i.e., thosewith the genomic signature of stem cells) maintain a high invasivecapacity by actively modulating their E-cadherin expression.

To further confirm E-cadherin expression changes during E-cad⁺ cellularinvasion, the time course of E-cadherin expression during invasion wascharacterized. Four hours after plating E-cad⁺ cells for invasionassays, cells on the top chamber were removed and the invaded cells (onthe underside of the membrane) were either stained immediately forE-cadherin (t=0) or incubated for additional times (5, 10 or 15 h) priorto staining (FIGS. 20A and 20B). Invaded E-cad⁺ DU145 and PC3 cellsinitially (t=0) did not express E-cadherin, but began to re-expressE-cadherin at 5 h and showed increasing E-cadherin expression thereafter(FIGS. 20A and 20B). These findings clearly support the notion thatduring the course of invasion the E-cad⁺ population can modulateE-cadherin expression in a time-dependent manner.

To study the molecular underpinnings of the E-cadherin modulationdisplayed by DU145 and PC3 E-cad⁺ cells plated in the top invasionchamber were analyzed at various times (0, 2, 4, 8, 16 and 24 h) forboth the expression of E-cadherin and its repressors Slug and Snail.Slug expression increased sharply at 2 hr in both cell lines (FIGS. 20Cand 20D); particularly in PC3 cells, which exhibited a nearly 20-foldincrease (FIG. 20D), and then declined at later times when cells wereobserved to invade. Invaded E-cad⁺ cells first appeared in the bottomchamber 4 hr after plating; t=0, (FIGS. 20A and 20B). In concert, theexpression of E-cadherin of cells in the top chamber decreased duringthe invasion period (FIGS. 20C and 20D). These findings imply that Slugabrogates E-cadherin transcription at early times in the invasionprocess of prostate cancer cells, a conclusion that is consistent withthe implication of Slug expression in the cellular proliferation andinvasion of PC3 cells.

Despite the pivotal role for E-cadherin in the invasion process, themodulation of surface E-cadherin expression was hypothesized to serve asa permissive factor for cells already capable of invading, and otherfactors were driving the invasive ability of E-cad⁺ cells. To test thispossibility, the effect of targeted knockdown of E-cadherin on theinvasion of parental DU145 and PC3 cells was examined. E-cad⁻ cells hadalready been observed to be non-invasive; therefore, E-cadherinknockdown would effectively target the E-cad⁺ cells. A reduction inE-cadherin expression, functionally mimicking Slug activity, wouldincrease invasion. In PC3 cells the opposite results were consistentlyobserved: siRNA-mediated E-cadherin knockdown cells (FIG. 21A, rightpanel) exhibited a lower invasive capacity compared to control cells(FIG. 21C and FIG. 22A). However, in this cell line efficient E-cadherinknockdown also resulted in markedly reduced levels of embryonic stemcell markers SOX2, OCT3/4 and c-Myc, as well as β-catenin, c-Met andNestin known to be involved in the ability of cell to act as stem cellsand in invasiveness (FIG. 21A, right panel). In contrast, in DU145cells, efficient E-cadherin knockdown (FIG. 21A, left panel) did notsignificantly reduce either cell invasion (FIG. 21B and FIG. 22A), orexpression of SOX2, OCT3/4, c-Myc, β-catenin and Nestin, except c-Met(FIG. 21A, left panel). These results suggest that if E-cadherinexpression could affect the expression of SOX2 and OCT3/4, as observedin PC3 cells (FIG. 21A, right panel), invasion would be impaired (FIG.21C). However, in the absence of efficient knockdown of these embryonicstem cell markers, E-cadherin knockdown did not significantly reducecellular invasion (FIG. 21A, left panel and FIG. 21B), demonstratingthat the down-regulation of E-cadherin alone is not sufficient forcellular invasion. Parenthetically, many studies have demonstrated thatE-cadherin expression may be associated with reduced invasion. However,the majority of these investigations utilized trypsin prior to cellplating, an enzyme which strips extracellular E-cadherin (FIG. 23) andeffectively reduces E-cadherin expression, perhaps similar to E-cadherinknockdown, thus confounding interpretation. Conversely, targetedknockdown of the embryonic stem cell factors SOX2 (FIG. 21D) or OCT3/4(FIG. 21G) resulted in reduced E-cadherin, β-catenin, c-Myc, c-Met andNestin levels and a statistically significant reduction in cellularinvasion (FIGS. 21E, 21F, 21H, and 21I; FIGS. 22B and 22C),demonstrating that the embryonic stem cell markers are required forinvasion. Therefore, to invade successfully, the prostate cancer stemcells must express the transcription factors SOX2 and OCT3/4 and mustalso possess the ability to express E-cadherin.

Example 10 Prostate Cancer Stem Cells Modulate Invasiveness byModulating E-Cadherin Expression

The process of Epithelial to Mesenchymal Transition, or EMT, isessential for development, and is an important part of neoplastictransformation. The EMT program, which involves the initiation of agenetic and epigenetic program resulting in the transition from anepithelial to a mesenchymal or fibroblastic phenotype, is a complexprocess that remains poorly understood. A process termed the CadherinSwitch, in which E-cadherin-expressing epithelial cells begin todown-regulate E-cadherin and up-regulate the mesenchymal cell markerN-cadherin, has been well documented during the EMT process in vitro. Alarge portion of the literature has examined the significance ofE-cadherin expression (FIG. 24A), and mounting evidence suggests thatE-cadherin expression is positively correlated with cancer patientprognosis.

Significantly, the above data indicated that the ability to modulateE-cadherin, rather than the absolute E-cadherin expression levels, maybe a more reliable indicator of cancer stem cells and invasiveness.Without being bound to a particular theory, it is proposed that theacquisition, or reacquisition, of E-cadherin protein expression in DU145and PC3 prostate cancer cells is a post-EMT process, and is required forthe progression to an invasive phenotype (FIG. 24B). E-cadherin ishighly present in various types of metastatic lesions, but the mechanismof E-cadherin re-expression in these cancer cells remains poorlyunderstood. Because the DU145 and PC3 cells exhibit a mixed EMTphenotype and are believed to have already undergone EMT, these cellsdisplay properties of both epithelial and mesenchymal cells and do notfit into the conventional EMT molecular profiles. Consistent with theseresults, the expression of mesenchymal markers Slug, Snail, and Vimentin(a critical marker for EMT) was observed not only in parental DU145 andPC3 cells (FIG. 25A) but also at high levels in E-cad⁻ compared toE-cad⁺ cells (FIGS. 25B and 25C). E-cad⁻ DU145 and PC3 cells representnon-invasive subpopulations, indicating that low E-cadherin expressionand high Slug, Snail and Vimentin expression are not sufficient to leadto an invasive phenotype. Rather, successful invasion was dependent onthe expression of the transcription factors SOX2 or OCT3/4, along withthe ability to modulate molecules related to EMT.

Characterization of the cancer stem cell population remains acontroversial issue. Because of the diverse etiologies of tumor typesthat arise in organs, the related cancer stem cell marker subset appearsto depend on the microenvironment in which these cells arise. Althoughmany studies have examined the cancer stem cell marker profiles derivedfrom primary and cultured tumor cell populations, little consensusexists concerning the definition of this elusive cell subpopulation. Theexpression of the embryonic stem cell markers SOX2, OCT3/4, and Nanogresults in a highly plastic, dedifferentiated, tumor-initiating stemcell phenotype. Cell surface markers including CD133 and CD44 have alsobeen used extensively, although their expression may be celltype-specific. While it is clear that enriched cancer stem-like cellpopulations form tumors with high efficiency when injected into SCIDmice, few studies have examined the invasive properties of these cells.It has been reported that CD133⁺ pancreatic cancer cells, CD44⁺ CD24⁻breast cancer cells, and CD44⁺ prostate cancer cells were more invasivethan their non-stem-like counterparts. The present study characterizedthe invasive ability of SOX2 and OCT3/4-expressing prostate cancer stemcells, and the role of E-cadherin modulation in this process.

A subset of the E-cadherin literature describes E-cadherin expression inmetastatic tissues and as a marker for tumor recurrence (FIG. 24A).E-cadherin expression is epigenetically silenced via promotermethylation in a large number of cancers, but E-cadherin is re-expressedin advanced prostate cancers and in prostate cancer metastases. Themechanism for the re-expression of E-cadherin in advanced disease andmetastases is not yet clear. The invention described herein is based onthe discovery of this mechanism reconciling early E-cadherin silencingand late-stage E-cadherin involvement in prostate cancer invasion.

The events described in the present study occurred after the EMT-likeprocess, and not as part of EMT itself. The post-EMT evolution of atumor into frank aggressive neoplasia appears to involve the emergenceof highly invasive E-cad⁺ cells. In addition to the expression ofembryonic stem cell markers SOX2 and OCT3/4 the ability of this tumorcell subpopulation to modulate E-cadherin expression should beconsidered as an indicator of prostate cancer stem cells. As such, theregulation of E-cadherin plasticity may provide targets for noveltherapies designed to interfere with the metastatic dissemination ofcancer stem cells.

The results reported above were obtained using the following methods andmaterials.

Human Embryonic and Prostate Cancer (PC) Cell Lines

Human metastatic prostate cancer cell lines were used in the studiesdescribed herein: DU145 (established from brain metastasis), LNCaP(established from lymph node metastasis) and PC3 (established from bonemetastasis). The human prostate cancer cell lines were obtained from theAmerican Type Culture Collection (ATCC; Manassas, Va.). Cells were grownin appropriate growth medium (ATCC) as suggested by ATCC. The humanembryonic stem cell line H9 was obtained from the National Stem CellBank and was cultured as described in Su et al. (Differentiation ofhuman embryonic stem cells into immunostimulatory dendritic cells underfeeder-free culture conditions. 2008. Clin. Cancer Res. 14:6207-6217).Clinical diagnoses were confirmed by the Department of Pathology at theUniversity of Florida. Human prostate cancer tissue microarrays werepurchased from Cybrdi.

Reagents

Commercially available PE-conjugated or FITC-conjugated monoclonal Abs(mAbs) against human CD9, CD24, CD44, E-cadherin, and mouse IgG1 isotypecontrol were used in the experiments described above (BD PharMingen; SanDiego, Calif.). Commercially available FITC-conjugated annexin V wasused in the experiments described above (BD PharMingen). Commerciallyavailable PE-conjugated mAbs against CD133 were used in the experimentsdescribed above (Miltenyi Biotech; Auburn, Calif.). Commerciallyavailable PE-conjugated or FITC-conjugated Abs against PODXL, SSEA1, andSSEA4 were used in the experiments described above (R&D Systems;Minneapolis, Minn.). Commercially available primary rabbit or mouse Absagainst β-actin, c-Myc, Klf4, Nanog, OCT3/4 and Sox2 were used in theexperiments described above (Santa Cruz Biotechnology; Santa Cruz,Calif.). Commercially available Agar Noble used in the experimentsdescribed above was obtained from Becton, Dickinson and Company (Sparks,Md.). Propidium iodide (PI) and crystal violet were obtained from SIGMA(St. Louis, Mo.).

Immunofluorescence

For immunofluorescence in the experiments described above, cells wereseeded on uncoated glass slides at approximately 2000 cells cm² andcultured for 4 days; cells were fixed at −20° C. in cold methanol for 8minutes and subsequently washed in phosphate-buffered saline (PBS).Enzyme treatment was not performed. Cells were stained with specific Abs(e.g., E-cadherin (BD Biosciences) or CD44 (Cell Signaling, Danvers,Mass.). Non-specific binding of the secondary Abs was reduced with anappropriate serum block. After staining, all slides were examined andpictures were taken using a commercially available fluorescencemicroscope (Carl Zeiss, Jena, Germany). Magnification of each picture isindicated as (×number).

Alternatively, cells were grown on glass coverslips, fixed in 4%paraformaldehyde (Sigma), and permeabilized with 0.2% Triton X-100/PBS.The cells were blocked with 10% goat serum/0.05% Triton X-100/PBS beforeincubating with commercially available primary antibodies(anti-β-cadherin, anti-OCT3/4 from Santa Cruz, anti-SOX2 from AbCam,anti-β-catenin from BD Bioscience; CD44 from Cell Signaling (Danvers,Mass.)) overnight at 4° C. The slides were washed, incubated withcommercially available Alexa Fluor 594— and/or Alexa Fluor488—conjugated secondary antibodies (Molecular Probes) and mounted usinga commerically available mounting medium (Vectashield; VectorLaboratories) containing DAPI to counterstain nuclei. The processedcells were examined using a a commercially available fluorescencemicroscope (Zeiss Axiophot microscope). Magnification of each picture isindicated as (×number).

Flow-Cytometry Analysis and Fluorescence-Activated Cell Sorting

Flow-cytometry in the experiments described above was performed bystandard methods. Flow-cytometry was used to analyze the expression ofcell surface molecules. Single cell suspensions were prepared bytrypsinization and then incubated in fresh medium on a rocker platformto enable regeneration of cell adhesion molecules. The cells werewashed, suspended in PBS containing 1% BSA and 1 mM CaCl2, and stainedwith commercially available primary antibodies for E-cadherin, PODXL,SSEA1, SSEA4 (R&D Systems), CD44, CD9, CD24, Integrin-α2β1 (BDPharmingen), ESA (Biomeda), and CD133 (Miltenyi Biotech). In addition,cell death was analyzed using FITC-conjugated annexin V and propidiumiodide (PI).

Analyses of fluorescence staining were performed using a commerciallyavailable flow cytometer (Becton Dickinson FACScan; San Jose, Calif.).Cells stained with propidium iodide (Sigma) were sorted using acommercially available Fluorescent-activated cell sorting (FACS) system(FACSCalibur flow cytometer, Becton Dickinson). E-cadherin positive andnegative cells were sorted by Fluorescent-activated cell sorting (FACS)analysis (Mo-Flo Cell Sorter; Becton-Dickinson). Live single cells weregated for analysis and sorted (FACSAriaSORP Cell Sorter with Diva 6.1software, Becton Dickinson).

Prostate Spheroid Culture

The prostate spheroid culture assay was performed according to themethod of Shi et al. (Anchorage-independent culture maintains prostatestem cells. 2007. Dev. Biol. 312:396-406). E-cadherin high- andlow-expressing cell subpopulations were collected after invasion, andspheroid formation assays were performed.

Soft Agar Assay

Cancer stem cells and non-cancer stem cells were isolated from DU145,LNCaP and PC3 cells. Cells were suspended in growth medium containing0.3% agar and layered over a 0.6% agar base layer to a final celldensity of 2×10³ cells/well. Cells were fed with fresh growth mediaevery 4-5 days for 2-3 weeks until the colonies were well formed. Cloneswere stained with 0.005% crystal violet for visualization.

Invasion Assay

Matrigel invasion assays were used according to the manufacturer'sinstructions (BD Biosciences, San Jose, Calif.). Cells were washed,resuspended in serum-free medium, and plated in the top chamber. Fetalbovine serum (FBS) was used as a chemoattractant in the bottom chamber.Chambers were incubated for 24 hr. Uninvaded cells (remaining in the topchamber) were removed with a cotton swab and invaded cells (at thebottom of the membrane) were fixed with 4% paraformaldehyde and stainedwith crystal violet. The membranes were mounted onto slides, and theinvaded cells were counted. To examine E-cadherin expression in top andbottom chambers, invasion chambers were used in parallel. Top-chambercells were stained with E-cadherin after removing invaded cells. Induplicate samples, the top chamber cells were removed, and the invadedcells were stained. For experiments examining E-cadherin re-expression,the chambers were incubated for 4 hr, and the top-chamber cells wereremoved. The invaded cells were incubated for an additional 5, 10 or 15hr. At the end of each incubation time, cells were fixed and stainedwith E-cadherin. For qPCR, top-chamber cells were collected bytrypsinization

Reverse Transcription Polymerase Chain Reaction (RT-PCR)

For the experiments described above, total RNA was extracted by using acommercially available kit (RNeasy Mini Kit; Qiagen, Valencia, Calif.),according to the manufacturer's instructions. Reverse transcriptionreactions were performed by standard methods using a a commerciallyavailable kit (Transcriptor First Strand cDNA Synthesis Kit; Roche,Indianapolis, Ind.). PCR was performed by standard methods using acommercially available Taq DNA polymerase (Roche) or commerciallyavailable PCR mix (GoTaq Green Master Mix, Promega). For the reactions,β-Actin transcript levels were used to normalize the amount of cDNA ineach sample. For the experiments described herein, primer sets used forRT-PCR analyses are listed in Table 2.

TABLE 2 List of primers for RT-PCR. annealing Gene name primer sequenceproduct size temperature GAPDH F5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′ 892 60°C. R5′-CATGTGGGCCATGAGGTCCACCAC-3′ B-actin F5′-CTCTTCCAGCCTTCCTTCCT-3′311 55° C. R5′-TCGTCATACTCCTGCTTGCT-3′ B-actinF5′-CAGCCATGTACGTTGCTATCCAGG-3′ 140 55° C.R5′-AGGTCCAGACGCAGGATGGCATG-3′ Oct3/4 F5′-ATTCAGCCAAACGACCATCT-3′ 37155° C./60° C. R5′-CAGCAGCCTCAAAATCCTCT-3′ Oct3/4 (4A)F5′-ACACCTGGCTTCGGATTTCGCCT-3′ 624 60° C.R5′-GCTTCCTCCACCCACTTCTGCAGC-3′ Sox2 F5′-CCCCCGGCGGCAATAGCA-3′ 448 55°C. R5′-TCGGCGCCGGGGAGATACAT-3′ Sox2 F5′-CGGAAAACCAAGACGCTCAT-3′ 445 55°C. R5′-TGGAGTGGGAGGAAGAGGTA-3′ cMyc F5′-TACCCTCTCAACGACAGCAG-3′ 468 55°C./60° C. R5′-TCTTGACATTCTCCTCGGTG-3′ Nanog F5′-TCTCCTCTTCCCTCCTCCAT-3′487 55° C./60° C. R5′-GGATGTTCTGGGTCTGGTTG-3′ Klf4F5′-GAGAGAGACCGAGGAGTTCA-3′ 480 55° C./60° C.R5′-CCTTTGCTGACGCTGATGAC-3′ Klf4#2 F5′-CAGCGACGCGCTGCTC-3′ 987 62° C.R5′-TGCAGGAACCGGGTGGCATG-3′ PSA F5′-GGTGACCAAGTTCATGCTGTG-3′ 195 60° C.R5′-GTGTCCTTGATCCACTTCCG-3′ AR F5′-GAAGCCATTGAGCCAGGTGT-3′ 164 60° C.R5′-TCGTCCACGTGTAAGTTGCG-3′ B-catenin F5′-ACTGGCAGCAACAGTCTTACC-3′ 83660° C. R5′-TCGTCCACGTGTAAG′TTGCG-3′ hE-cadherinF5′-GAACGCATTGCCACATACACT-3′ 745 60° C. R5′-CTGTGGAGGTGGTGAGAGAGA-3′tert F5′-GCACGGCTTTTGTTCAGATG-3′ 407 55° C. R5′-GTTCTTGGCTTTCAGGATGG-3′β-microglobulin F5′-AGCGTACTCCAAAGATTCAGGTT-3′ 60° C.R5′-TACATGICTCGATCCCACTTAACTAT-3′ uPAR F5′-CGTGAGCTGGTGGAGAAAAG-3′ 60°C. R5′-TGTTGCAGCATTTCAGGAAG -3′ Keratin 8 F5′-TGAGGTCAAGGCACAGTACG -3′60° C. R5′-TGATGTTCCGGTTCATCTCA -3′

Semi-quantitative RT-PCR was also performed by using a panel offirst-strand cDNAs from 48 human prostate samples (TissueScan ProstateCancer II, Origene). H9 cells, and the primary prostate stem-cell likeline were analyzed and normal human prostate RNA (Clontech) was used asa control. Relative transcript levels were normalized to β-actin levelsfor each case. In order to detect pseudogenes of Oct4A, the procedure ofPanagopoulos et. al. 2008. Genes Chromosomes Cancer 47:521-529) wasused.

For real-time PCR experiments involving EMT-related genes, total RNA wasisolated with RNAqueous-Micro (Ambion, Austin, Tex.). Reversetranscription was performed with the Verso cDNA Kit (Thermo Scientific,Waltham, Mass.) according to the manufacturer's recommendations.Quantitative real-time PCR (qPCR) was performed to determine theexpression levels of EMT-related genes (E-cadherin, Slug, Snail, andVimentin). Primers (1 μl), SYBR Green PCR Master Mix (AppliedBiosystems, Carlsbad, Calif.) and cDNA (20 ng) reaction mixture wasperformed on the ABI 7500 Fast Real-Time PCR System (AppliedBiosystems). The sequences of the primers are listed below:

E-cadherin Forward, 5′-ACCAGAATAAAGACCAAGTGACCA-3′ E-cadherin Reverse,5′-AGCAAGAGCAGCAGAATCAGAAT-3′ Slug Forward,5′-GAGTCTGTAATAGGATTTCCCATAG-3′ Slug Reverse,5′-CTTTAGTTCA ACAATGGCAAC-3′ Snail Forward, 5′-TTGGATACAGCTGCT TTGAG-3′Snail Reverse, 5′-ATTG CATAGTTAGTCACACCTC-3′ Vimentin Forward,5′-AATGGCTCGTCACCTTCGT GAAT-3′ Vimentin Reverse,5′-CAGATTAGTTTCCCTCAGGTTCAG-3′ Actin Forward,5′-CTCCTCC TGAGCGCAAGTACTC-3′ Actin Reverse,5′-TCCTGC TTGCTGATCCACATC-3′.The mRNA expression was normalized to actin according to the ΔΔCtmethod. Gene expression of the initiating (t=0) samples was defined as“1”.

Histological and Immunohistochemical Analysis

For the experiments described above, commercially available tissuearrays (Cybrdi) were used. Additional tissues were obtained from theDepartment of Urology at the University of Florida. These were processedas described in Gibbs et al. (Stem-like cells in bone sarcomas:implications for tumorigenesis. 2005. Neoplasia 7:967-976) using eithera commercially available OCT3/4 (AbCam) antibody or a commerciallyavailable SOX2 (R&D Systems) antibody. To assess nuclear staining, anarbitrary system was used by a pathologist blinded to sample identity.Twenty random fields were examined and the overall percentage ofpositive nuclear staining was histologically scored.

Western-Blot Analysis

For the experiments described above, Western-blot analysis was performedby standard methods on isolated cancer stem cells and non-cancer stemcells. Whole cell lysates were prepared in lysis buffer with acommercially available protease inhibitor cocktail (Pierce) at 4° C.,followed by centrifugation at 13,000×g for 10 min. Extracts wereseparated by SDS/PAGE and transferred to nitrocellulose membranes. Themembranes were blocked with 5% nonfat dry milk in 20 mM Tris-HCl (pH7.5), 500 mM sodium chloride, and 0.05% Tween 20 for 2 h and thenincubated with commercially available primary antibodies in the samebuffer with 1% BSA (fraction V). Primary antibodies against OCT3/4,c-Myc, c-Met, and β-actin (Cell Signaling), OCT3/4, SOX2, Klf4, andtubulin (Santa Cruz Biotechnology), Nanog (BioLegend), and Nestin,β-catenin, and E-cadherin (BD Bioscience) were used in these studies.After washing, the blots were incubated with an HRP-conjugated secondaryantibody and visualized with a commercially available chemiluminescencedetection system (Amersham). β-actin-specific Abs were used to ensureequal protein loading.

Mouse Xenograft Model of Human PC

For the experiments described above, male C.B-17/IcrHsd SCID mice, 5 to6 weeks old, were obtained from Harlan Sprague Dawley (Indianapolis,Ind.). To examine tumorigenicity, sorted cells from PC3 and DU145 cellswere injected subcutaneously into groups of mice (5 mice per group) at adose of 1×10³ PC3 cells/mouse or 1×10⁵ DU145 cells/mouse; andexperiments repeated twice. Experiments were performed under an approvedprotocol of the Institutional Animal Care and Use Committee of theUniversity of Florida. Animals were monitored, and tumor size wasmeasured twice a week. Mice were humanely sacrificed when moribund orwhen subcutaneous tumors reached 15 mm in diameter.

siRNA and shRNA Transfection

For the experiments described above, commercially available c-Myc, Klf4,Nanog, OCT3/4, Sox2 and control siRNAs, transfection reagent, andtransfection medium (Santa Cruz Biotechnology) were used. Gene silencingof specific target genes was performed according to the manufacturer'sprotocol. Control siRNA was also used for these experiments.

For OCT3/4 or SOX2 shRNA knock-down experiments, commercially availableplasmid vectors encoding either OCT3/4 or SOX2 were used (Origene). Fortransfection, 1.5×10⁵ DU145 cells/well were seeded in 6 well plates inmedia without antibiotics the day before the experiment. Cells werewashed with buffer (Optimem, Invitrogen) and then transfected using acommercially available transfection reagent (Lipofectamine, Invitrogen).Transfected cells were selected using puromycin, pooled, and single-cellcloned before Western blot analysis for OCT3/4 or SOX2 expression.

For E-cadherin shRNA knock-down experiments, shRNA-mediated knockdownwas performed. E-cadherin SmartPool siRNAs (Dharmacon, Lafayette, Colo.)were transfected into cells using the DharmaFECT 1 reagent (Dharmacon)or Oligofectamine (Invitrogen, Carlsbad, Calif.) according to themanufacturers' instructions. After 72 hr, cells were analyzed asindicated.

Statistical Analysis

Student t test was used for the comparison of various experimentalgroups. Significance was set at P<0.05. One-way ANOVA, Newman Keulstesting and Spearman coefficient of rank correlation were calculatedusing Prism version 4 (GraphPad Software, Inc.). Significance was set atp<0.05. Results indicated by an asterisk (*) were considered to bestatistically significant.

Other Embodiments

From the foregoing description, it will be apparent that variations andmodifications may be made to the invention described herein to adopt itto various usages and conditions. Such embodiments are also within thescope of the following claims.

The recitation of a listing of elements in any definition of a variableherein includes definitions of that variable as any single element orcombination (or subcombination) of listed elements. The recitation of anembodiment herein includes that embodiment as any single embodiment orin combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are hereinincorporated by reference to the same extent as if each independentpatent and publication was specifically and individually indicated to beincorporated by reference.

1. A method for identifying a neoplasia in a subject, the methodcomprising identifying an increased level of a Marker nucleic acidmolecule or polypeptide selected from the group consisting of OCT3/4,Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR in a biological samplederived from the subject, relative to the level present in a reference,thereby identifying a neoplasia in the subject.
 2. The method of claim1, wherein the neoplasia is prostate carcinoma in the subject.
 3. Themethod of claim 1, wherein the identification of increased levels ofOCT3/4, Nanog, and Sox2 nucleic acid molecules or polypeptides in abiological sample derived from the subject, relative to the levelspresent in a reference, identifies a neoplasia in the subject.
 4. Themethod of claim 1, further comprising identifying an increase in c-Mycnucleic acid molecules or polypeptides in a biological sample derivedfrom the subject, relative to the level present in a reference, therebyidentifying a neoplasia in the subject.
 5. The method of claim 1,wherein the Marker is Sox2.
 6. The method of claim 1, further comprisingidentifying an increase in E-cadherin nucleic acid molecules orpolypeptides in a biological sample derived from the subject, relativeto the level present in a reference, thereby identifying a neoplasia orprostate carcinoma in the subject.
 7. The method of claim 1, wherein thebiological sample is a tissue sample or biopsy sample.
 8. The method ofclaim 1, wherein the biological sample is a biological fluid selectedfrom the group consisting of blood, blood serum, plasma, saliva, urine,seminal fluid, and ejaculate.
 9. The method of claim 1, wherein anincrease in the level of one or more of said Markers distinguishesprostate carcinoma from benign prostatic hyperplasia.
 10. A method fordiagnosing the presence or absence of neoplasia in a subject, the methodcomprising detecting the level of OCT3/4, Nanog, and Sox2 Markerpolypeptides or nucleic acid molecules in blood of a subject, relativeto the level present in a reference, wherein detection of an increase insaid Markers diagnoses the subject as having neoplasia, and failure todetect said Markers diagnoses the subject as not having neoplasia. 11.The method of claim 1, where the neoplasia is prostate cancer.
 12. Themethod of claim 10, further comprising detecting a c-Myc nucleic acidmolecule or polypeptide in blood of the subject, relative to the levelpresent in a reference, wherein an increased level of c-Myc identifiesthe subject as having neoplasia or prostate cancer.
 13. (canceled)
 14. Amethod for identifying a prostate carcinoma in a subject, the methodcomprising a) isolating cells that bind an E-cadherin capture reagentfrom a biological sample derived from a subject; and b) analysing saidcells for a Marker nucleic acid molecule or polypeptide selected fromthe group consisting of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, anduPAR, wherein an increased level of said Marker relative to the levelpresent in a reference, identifies a prostate carcinoma in the subject.15-16. (canceled)
 17. A method for characterizing the aggressiveness ofa prostate cancer in a subject, the method comprising comparing thelevel of a one or more nucleic acid molecules or polypeptide Markersselected from the group consisting of OCT3/4, Nanog, Sox2, c-Myc, Klf4,Keratin 8, and uPAR in a biological sample of the subject, relative tothe level present in a reference, wherein an increase in the level ofone or more of said Markers identifies the prostate cancer as aggressiveand the absence of an increase identifies the prostate cancer as lessaggressive. 18-22. (canceled)
 23. The method of claim 17, wherein anincrease in the level of one or more of said Markers identifies theprostate cancer as metastatic or as having a propensity to metastasize.24-33. (canceled)
 34. The method of claim 17, wherein an increase in thelevel of one or more of said Markers identifies the subject as having apoor prognosis. 35-38. (canceled)
 39. A method of monitoring prostatecancer therapy in a subject, the method comprising determining the levelof a Marker selected from the group consisting of OCT3/4, Nanog, Sox2,c-Myc, Klf4, Keratin 8, and uPAR nucleic acid molecule or polypeptide ina biological sample derived from the subject, relative to the levelpresent in a reference, wherein a prostate cancer therapy that reducesthe level of said marker is identified as effective. 40-44. (canceled)45. A method of selecting a treatment for a subject diagnosed as havingprostate cancer, the method comprising: (a) detecting the presence orabsence of one or more markers selected from the group consisting ofOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR in a biologicsample from the subject; and (b) selecting a treatment from the groupconsisting of surveillance, surgery, hormone therapy, chemotherapy, andradiotherapy. 46-49. (canceled)
 50. The method of claim 1, wherein thebiological sample is a tissue sample selected from the list consistingof prostate tissue and peripheral blood mononuclear cells (PBMC) orwherein the biological sample is a biological fluid selected from thegroup consisting of blood, blood serum, plasma, saliva, urine, seminalfluids, and ejaculate. 51-61. (canceled)
 62. A kit for the analysis ofOCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPAR, the kitcomprising at least one agent capable of specifically binding orhybridizing to an OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, anduPARpolypeptide or nucleic acid molecule Markers, and directions forusing the agent for the analysis of OCT3/4, Nanog, Sox2, c-Myc or Klf4Markers. 63-68. (canceled)
 69. A microarray comprising Markers bound toa solid support, wherein the Markers are selected from the groupconsisting of OCT3/4, Nanog, Sox2, c-Myc, Klf4, Keratin 8, and uPARpolypeptides or nucleic acid molecules, or fragments thereof. 70-71.(canceled)